Laccases and methods of use thereof at low temperature

ABSTRACT

Laccase enzymes and nucleic acid sequences encoding such laccase enzymes are described. The laccase enzymes may be employed in conjunction with mediators in improved methods for modifying the color of denim fabrics. Low temperature and single-bath textile processing using laccase enzymes are also described.

PRIORITY

The present application claims priority to U.S. Provisional PatentApplication Ser. Nos. 61/140,724, filed on Dec. 24, 2008, 61/154,882,filed on Feb. 24, 2009, and 61/237,532, filed on Aug. 27, 2009, each ofwhich is incorporated by reference in its entirety.

TECHNICAL FIELD

The present systems, compositions, and methods relate to laccase enzymesand nucleic acid sequences encoding such laccase enzymes. The laccaseenzymes may be employed in conjunction with mediators in improvedmethods for modifying the color of denim fabrics.

BACKGROUND

Laccases are copper-containing phenol oxidizing enzymes that are knownto be good oxidizing agents in the presence of oxygen. Laccases arefound in microbes, fungi, and higher organisms. Laccase enzymes are usedfor many applications, including pulp and paper bleaching, treatment ofpulp waste water, de-inking, industrial color removal, bleaching inlaundry detergents, oral care teeth whiteners, and as catalysts orfacilitators for polymerization and oxidation reactions.

Laccases can be utilized for a wide variety of applications in a numberof industries, including the detergent industry, the paper and pulpindustry, the textile industry and the food industry. In oneapplication, phenol oxidizing enzymes are used as an aid in the removalof stains, such as food stains, from clothes during detergent washing.Most laccases exhibit pH optima in the acidic pH range while beinginactive in neutral or alkaline pHs.

Laccases are known to be produced by a wide variety of fungi, includingspecies of the genii Aspergillus, Neurospora, Podospora, Botrytis,Pleurotus, Fornes, Phlebia, Trametes, Polyporus, Stachybotrys,Rhizoctonia, Bipolaris, Curvularia, Amerosporium, Lentinus,Myceliophtora, Coprinus, Thielavia, Cerrena, Streptomyces, andMelanocarpus. However, there remains a need for laccases havingdifferent performance profiles in various applications.

For many applications, the oxidizing efficiency of a laccase can beimproved through the use of a mediator, also known as an enhancingagent. Systems that include a laccase and a mediator are known in theart as laccase-mediator systems (LMS). The same compounds can also beused to activate or initiate the action of laccase.

There are several known mediators for use in a laccase-mediator system.These include HBT (1-hydroxybenzotriazole), ABTS[2,2′-azinobis(3-ethylbenzothiazoline-6-sulfinic acid)], NHA(N-hydroxyacetanilide), NEIAA (N-acetyl-N-phenylhydroxylamine), HBTO(3-hydroxy 1,2,3-benzotriazin-4(3H)-one), and VIO (violuric acid). Inaddition, there are several compounds containing NH—OH or N—O groupsthat have been found to be useful as mediators.

Functional groups and substituents have large effects on mediatorefficiency. Even within the same class of compounds, a substituent canchange the laccase specificity towards a substrate, thereby increasingor decreasing mediator efficacy greatly. In addition, a mediator may beeffective for one particular application but unsuitable for anotherapplication. Another drawback for current mediators is their tendency topolymerize during use. Thus, there is a need to discover efficientmediators for specific applications. One such application is thebleaching of textiles, wherein it is also important that the mediatorsare not unduly expensive or hazardous. Other applications of thelaccase-mediator system are given below.

Methods of use for laccases at low temperatures would provide a benefitin terms of energy savings, for example, in textile processing methodswhere energy input for heating of processing baths could be reduced.Development of methods in which laccase enzymes are used at lowtemperatures for applications such as enzymatic bleaching would bedesirable.

SUMMARY

Described are enzymatic oxidation systems, compositions, and methods,involving laccases. In one aspect, a textile processing method isprovided, comprising contacting a textile with a laccase enzyme and,optionally, a mediator at a temperature less than 40° C., for a lengthof time and under conditions sufficient to cause a color modification ofthe textile. In some embodiments, the color modification is selectedfrom lightening of color, change of color, change in color cast,reduction of redeposition/backstaining, and bleaching. In someembodiments, the temperature is from about 20° C. to less than 40° C. Insome embodiments, the temperature is from about 20° to about 35° C. Insome embodiments, the temperature is from about 20° C. to about 30° C.In some embodiments, the temperature is from about 20° C. to about 23°C. In some embodiments, the temperature is 20° C., 21° C., 22° C., 23°C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32°C., 33° C., 34° C., or 35° C. In some embodiments, the temperature isthe ambient temperature of tap water.

In some embodiments, the textile is indigo-dyed denim In someembodiments, the textile is sulfur-dyed denim In some embodiments, thedenim is desized and/or stonewashed prior to or simultaneously withcontacting the textile with the laccase enzyme and the mediator. In someembodiments, the stonewashing and contacting the textile with thelaccase enzyme and the mediator occur in the same bath.

In some embodiments, the method further comprises contacting the textilewith a cellulase enzyme, simultaneously or sequentially with contactingthe textile with the laccase enzyme and the mediator. In someembodiments, contacting the textile with the cellulase enzyme andcontacting the textile with the laccase enzyme and the mediator areperformed sequentially, and wherein contacting the textile with thecellulase enzyme is performed prior to contacting the textile with thelaccase enzyme and the mediator. In some embodiments, contacting thetextile with the cellulase enzyme and contacting the textile with thelaccase enzyme and the mediator are performed sequentially in the samebath without draining the bath between contacting the textile with acellulase enzyme and contacting the textile with the laccase enzyme andthe mediator.

In some embodiments, contacting the textile with the cellulase enzymeand contacting the textile with the laccase enzyme and the mediator areperformed a temperature less than 40° C. In some embodiments, thetemperature is from about 20° C. to less than 40° C. In someembodiments, the temperature is from about 20° to about 35° C. In someembodiments, the temperature is from about 20° C. to about 30° C. Insome embodiments, the temperature is from about 20° C. to about 23° C.In some embodiments, the temperature is 20° C., 21° C., 22° C., 23° C.,24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C.,33° C., 34° C., or 35° C. In some embodiments, the temperature is theambient temperature of tap water.

In some embodiments, the cellulase enzyme acts synergistically with thelaccase enzyme to produce a textile with a greater degree of lighteningof color of the textile, change in color, change in color cast,reduction of redoposition/backstaining, and/or bleaching. In someembodiments, the cellulase enzyme acts additively with the laccaseenzyme to produce a textile with a greater degree of lightening of colorof the textile, change in color, change in color cast, reduction ofredoposition/backstaining, and/or bleaching in comparison to anidentical method in which cellulase is not included.

In some embodiments, the laccase is a microbial laccase. In someembodiments, laccase is from a Cerrena species. In some embodiments, thelaccase is from Cerrena unicolor. In some embodiments, the laccase islaccase D from C. unicolor.

In some embodiments, the laccase has an amino acid sequence that is atleast 70% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 19, and SEQ ID NO: 20. In some embodiments, the laccasehas an amino acid sequence that is at least 80% identical to an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.In some embodiments, the laccase has an amino acid sequence that is atleast 90%, or even at least 95%, identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

In some embodiments, the laccase has an amino acid sequence that is atleast 70% identical to SEQ ID NO: 19 or SEQ ID NO: 20. In someembodiments, the laccase has an amino acid sequence that is at least 80%identical to SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, thelaccase has an amino acid sequence that is at least 90% identical to SEQID NO: 19 or SEQ ID NO: 20. In some embodiments, the laccase has anamino acid sequence that is at least 95% identical to SEQ ID NO: 19 orSEQ ID NO: 20.

In some embodiments, the laccase enzyme and the mediator are providedtogether in a ready-to-use composition. In some embodiments, the laccaseenzyme and the mediator are provided in a solid form. In someembodiments, the laccase enzyme and the mediator are provided asgranules. In particular embodiments, the mediator is syringonitrile.

In another aspect, laccases, nucleic acid sequences encoding suchlaccases, and vectors and host cells for expressing the laccases areprovided. The laccases can be used at low temperatures in methods inwhich a reduction of energy input would be desirable, such as textileprocessing. In some embodiments, the laccase enzyme comprises, consistsof, or consists essentially of the amino acid sequence depicted in anyof SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, or 20, or an aminoacid sequence having at least about 60%, 65%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5%, identical to any ofSEQ ID NOs: 2, 4, 6, 8, 12, 14, 16, 18, 19, or 20. In particularembodiments, the laccase has an amino acid sequence that is at least70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even99.5%, identical to SEQ ID NO: 19 or SEQ ID NO: 20. In still moreparticular embodiments, the laccase has the amino acid sequence SEQ IDNO: 19 or SEQ ID NO: 20. Preferably, such polypeptides have laccaseenzymatic activity, which can be determined, e.g., using the assaysdescribed, herein.

In another aspect, a composition comprising a laccase enzyme comprising,consisting of, or consisting essentially of any of the aforementionedamino acid sequences is provided. In some embodiments, the compositionfurther comprises a buffering system to maintain the pH of thecomposition at about 5.5 to about 6.5 in solution. In some embodiments,the composition further comprises a mediator. The mediator may beselected from, e.g., acetosyringone, syringaldehyde, syringamide, methylsyringamide, 2-hydroxyehyl syringamide, methyl syringate,dimethylsyringamide, shrine acid, and4-hydroxy-3,5-dimethoxybenzonitrile (syringonitrile). In one embodiment,the mediator is 4-hydroxy-3,5-dimethoxybenzonitrile. In someembodiments, the composition is in a solid form. In some embodiments,the laccase enzyme and the mediator are provided together in aready-to-use composition. In some embodiments, the laccase enzyme andthe mediator are provided in a solid form. In some embodiments, thelaccase enzyme and the mediator are provided as granules. In particularembodiments, the mediator is syringonitrile.

In some embodiments, the laccase enzyme is used at a pH of about 5 toabout 7, a temperature of about 20° C. to about 30° C., a liquor ratioof about 5:1 to about 10:1, and for a time period of about 15 to about60 minutes.

These and other aspects and embodiments of the present system,compositions, and methods will be apparent from the description andaccompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effects of modifying the color of stonewashed denimwith laccase enzymes at different temperatures, as described in Example1.

FIG. 2 shows the effects of laccase and mediator ratios on modifying thecolor of stonewashed denim, as described in Example 2.

FIG. 3 shows the effect of temperature on modifying the color ofstonewashed denim with a “ready to use” laccase composition, asdescribed in Example 3.

FIG. 4 shows the effect of temperature on color-modifying performance oflaccase enzymes on stonewashed denim, as described in Example 3.

FIG. 5 shows the effect of cellulase treatment in combination withlaccase-mediated color modification, as described in Examples 4-6.

DETAILED DESCRIPTION

Described are enzymatic oxidation systems, compositions, and methods,involving laccases. The systems, compositions, and methods are useful,for example, for low-temperature processing of textiles to affect colormodification. Such processing uses less energy than conventional textileprocessing technologies, and involves more environmentally-friendlychemical reagents. Various aspects and embodiments of the systems,compositions, and methods are to be described.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art. Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), andHale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, HarperPerennial, N.Y. (1991) provide a general dictionary of many of the termsused herein. The following terms are defined for additional clarity.

As used herein, the term “enzyme” refers to a protein that catalyzes achemical reaction. The catalytic function of an enzyme constitutes its“enzymatic activity” or “activity.” An enzyme is typically classifiedaccording to the type of reaction it catalyzes, e.g., oxidation ofphenols, hydrolysis of peptide bonds, incorporation of nucleotides, etc.

As used herein, the term “substrate” refers to a substance (e.g., achemical compound) on which an enzyme performs its catalytic activity togenerate a product.

As used herein, a “laccase” is a multi-copper containing oxidase (EC1.10.3.2) that catalyzes the oxidation of phenols, polyphenols, andanilines by single-electron abstraction, with the concomitant reductionof oxygen to water in a four-electron transfer process.

As used herein, “variant” proteins encompass related and derivativeproteins that differ from a parent/reference protein by a small numberof amino acid substitutions, insertions, and/or deletions. In someembodiments, the number of different amino acid residues is any of about1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments,variants differ by about 1 to about 10 amino acids residues. In someembodiments, variant proteins have at least about 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or even 99.5% amino acid sequence identity to aparent/reference protein.

As used herein, the term “analogous sequence” refers to a polypeptidesequence within a protein that provides a similar function, tertiarystructure, and/or conserved residues with respect to a sequence within aparent/reference protein. For example, in structural regions thatcontain an alpha helix or a beta sheet structure, replacement amino acidresidues in an analogous sequence maintain the same structural feature.In some embodiments, analogous sequences result in a variant proteinthat exhibits a similar or improved function with respect to the parentprotein from which the variant is derived.

As used herein, a “homologous protein” or “homolog” refers to a protein(e.g., a laccase enzyme) that has a similar function (e.g., enzymaticactivity) and/or structure as a reference protein (e.g., a laccaseenzyme from a different source). Homologs may be from evolutionarilyrelated or unrelated species. In some embodiments, a homolog has aquaternary, tertiary and/or primary structure similar to that of areference protein, thereby potentially allowing for replacement of asegment or fragment in the reference protein with an analogous segmentor fragment from the homolog, with reduced disruptiveness of structureand/or function of the reference protein in comparison with replacementof the segment or fragment with a sequence from a non-homologousprotein.

As used herein, “wild-type,” “native,” and “naturally-occurring”proteins are those found in nature. The terms “wild-type sequence”refers to an amino acid or nucleic acid sequence that is found in natureor naturally occurring. In some embodiments, a wild-type sequence is thestarting point of a protein engineering project, for example, productionof variant proteins.

As used herein, a “signal sequence” refers to a sequence of amino acidsbound to the N-terminal portion of a protein, and which facilitates thesecretion of the mature form of the protein from the cell. The matureform of the extracellular protein lacks the signal sequence which iscleaved off during the secretion process.

As used herein, the term “culturing” refers to growing a population ofmicrobial cells under suitable conditions in a liquid, semi-solid, orsolid medium for expressing a polypeptide of interest. In someembodiments, culturing is conducted in a vessel or reactor, as known inthe art.

As used herein, the term “derivative” refers to a protein that isderived from a parent/reference protein by addition of one or more aminoacids to either or both the N- and C-terminal end(s), substitution ofone or more amino acid residues at one or a number of different sites inthe amino acid sequence, deletion of one or more amino acid residues ateither or both ends of the protein or at one or more sites in the aminoacid sequence, and/or insertion of one or more amino acids at one ormore sites in the amino acid sequence. The preparation of a proteinderivative is often achieved by modifying a DNA sequence which encodesfor the native protein, transformation of that DNA sequence into asuitable host, and expression of the modified DNA sequence to form thederivative protein.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

As used herein, the term “expression vector” refers to a DNA constructcontaining a DNA coding sequence (e.g., gene sequence) that is operablylinked to one or more suitable control sequence(s) capable of effectingexpression of the coding sequence in a host. Such control sequencesinclude a promoter to effect transcription, an optional operatorsequence to control such transcription, a sequence encoding suitablemRNA ribosome binding sites, and sequences which control termination oftranscription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may, in some instances, integrate into the genomeitself.

As used herein, the term “host cell” refers to a cell or cell line intowhich a recombinant expression vector for production of a polypeptidemay be transfected, transformed, or otherwise introduced for expressionof a polypeptide. Host cells include progeny of a single host cell, andthe progeny may not necessarily be identical (in morphology or in totalgenomic DNA complement) to the parent cell due to natural, accidental,or deliberate mutation. A host cell may be bacterial or fungal. A hostcell includes a cell transfected or transformed in vivo with anexpression vector.

As used herein, the term “introduced,” in the context of inserting anucleic acid sequence into a cell includes “transfection,”“transformation,” and “transduction,” and refers to the incorporation ofa nucleic acid sequence into a eukaryotic or prokaryotic cell, whereinthe nucleic acid sequence is incorporated into the genome of the cell(e.g., chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed.

As used herein, “cleaning compositions” and “cleaning formulations”refer to compositions that may be used for the removal of undesiredcompounds from items to be cleaned, such as fabrics, dishes, contactlenses, hair (shampoos), skin (soaps and creams), teeth (mouthwashes,toothpastes), and other solid and surfaces. The terms encompass anymaterials/compounds selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, or spray composition), as long as the composition is compatiblewith the enzyme(s) used in the composition. The specific selection ofcleaning composition materials are readily made by considering thesurface, item or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use.

The terms further refer to any composition that is suitable forcleaning, bleaching, disinfecting, and/or sterilizing a object and/orsurface. It is intended that the terms include, but are not limited todetergent compositions (e.g., liquid and/or solid laundry detergents andfine fabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the terms “cleaning compositions” and “cleaning formulations”include (unless otherwise indicated) granular or powder-form all-purposeor heavy-duty washing agents, especially cleaning detergents; liquid,gel or paste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures that are intended for usein a wash medium for the cleaning of soiled objects. In someembodiments, the term is used in reference to laundering fabrics and/orgarments (e.g., “laundry detergents”). In alternative embodiments, theterm refers to other detergents, such as those used to clean dishes,cutlery, etc. (e.g., “dishwashing detergents”). In addition toenzyme(s), “detergent compositions” and “detergent formulations”encompasses detergents that contain surfactants, builders, bleachingagents, bleach activators, bluing agents and fluorescent dyes, cakinginhibitors, masking agents, enzyme activators, antioxidants, andsolubilizers.

As used herein, the phrase “detergent stability” refers to the stabilityof an enzyme, and optionally an associated substrate or mediator, in adetergent composition. In some embodiments, the stability is assessedduring the use of the detergent, while in other embodiments, the termrefers to the stability of a detergent composition during storage.

As used herein the term “hard surface cleaning composition,” refers todetergent compositions for cleaning hard surfaces such as floors, walls,tiles, stainless steel vessels (e.g., fermentation tanks), bath andkitchen fixtures, and the like. Such compositions may be provided in anyform, including but not limited to solids, liquids, emulsions, and thelike.

As used herein, the term “dishwashing composition” refers to all formsof compositions for cleaning dishes, including but not limited togranular and liquid forms.

As used herein, the term “disinfecting” refers to the removal or killingof microbes, including fungi, bacteria, spores, and the like.

As used herein, the term “fabric cleaning composition” refers a form ofdetergent composition for cleaning fabrics, including but not limitedto, granular, liquid and bar forms.

As used herein, the terms “polynucleotide,” “nucleic acid,” and“oligonucleotide,” are used interchangeably to refers to a polymericform of nucleotides of any length and any three-dimensional structure,whether single- or multi-stranded (e.g., single-stranded,double-stranded, triple-helical, etc.), which containdeoxyribonucleotides, ribonucleotides, and/or analogs or modified formsof deoxyribonucleotides or ribonucleotides, including modifiednucleotides or bases or their analogs. Because the genetic code isdegenerate, more than one codon may be used to encode a particular aminoacid. Any type of modified nucleotide or nucleotide analog may be used,so long as the polynucleotide retains the desired functionality underconditions of use, including modifications that increase nucleaseresistance (e.g., deoxy, 2′-O-Me, phosphorothioates, etc.). Labels mayalso be incorporated for purposes of detection or capture, for example,radioactive or nonradioactive labels or anchors, e.g., biotin. The termpolynucleotide also includes peptide nucleic acids (PNA).Polynucleotides may be naturally occurring or non-naturally occurring. Asequence of nucleotides may be interrupted by non-nucleotide components.One or more phosphodiester linkages may be replaced by alternativelinking groups. For example, phosphate may be replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, COor CH₂ (“formacetal”), in which each R or R′ is independently H orsubstituted or unsubstituted alkyl (1-20 C) optionally containing anether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl.Not all linkages in a polynucleotide need be identical. Polynucleotidesmay be linear or circular or comprise a combination of linear andcircular portions.

As used herein, the terms “polypeptide, “protein,” and “peptide,” referto a composition comprised of amino acids (i.e., amino acid residues).The conventional one-letter or three-letter codes for amino acidresidues are used. A polypeptide may be linear or branched, may comprisemodified amino acids, and may be interrupted by non-amino acids. Theterms also encompass an amino acid polymer that has been modifiednaturally or by intervention; for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent. Also included within the definition are, for example,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), as well as othermodifications known in the art.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally, e.g., as in a purified restriction fragment, orproduced synthetically, which is capable of acting as a point ofinitiation of nucleic acid synthesis when incubated with a complementarynucleic acid in the presence of nucleotides and polymerase at a suitabletemperature and pH. The primer is preferably single stranded but mayalternatively be double stranded. If double stranded, the primer isfirst treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The exact lengths of the primers will dependon many factors, including temperature, source of primer and the use ofthe method.

As used herein, the terms “recovered,” “isolated,” “purified,” and“separated” refer to a material (e.g., a protein, nucleic acid, or cell)that is removed from at least one component with which it isnaturally-associated, or associated as the result of heterologousexpression.

As used herein, the term “textile(s)” refers to fibers, yarns, fabrics,garments, and non-woven materials. The term encompasses textiles madefrom natural and synthetic (e.g., manufactured) materials, as well asnatural and synthetic blends. The term “textile” refers to bothunprocessed and processed fibers, yarns, woven or knit fabrics,non-wovens, and garments. In some embodiments, a textile containscellulose.

As used herein, the phrase “textile(s) in need of processing” refers toa textile that needs to be desized, scoured, bleached, and/orbiopolished to produce a desired effect.

As used herein, the phrase “textile(s) in need of color modification”refers to a textile that needs to be altered with respect to it color.These textiles may or may not have been already subjected to othertreatments. Similarly, these textiles may or may not need subsequenttreatments.

As used herein, the term “fabric” refers to a manufactured assembly offibers and/or yarns that has substantial surface area in relation to itsthickness and sufficient cohesion to give the assembly useful mechanicalstrength.

As used herein, the term “color modification” refers to a change in thechroma, saturation, intensity, luminance, and/or tint of a colorassociated with a fiber, yarn, fabric, garment, or non-woven material,collectively referred to as textile materials. Without being limited toa theory, it is proposed that color modification results from themodification of chromaphores associated with a textile material, therebychanging its visual appearance. The chromophores may benaturally-associated with the material used to manufacture a textile(e.g., the white color of cotton) or associated with special finishes,such as dying or printing. Color modification encompasses chemicalmodification to a chromophore as well as chemical modification to thematerial to which a chromophore is attached. Examples of colormodification include fading, bleaching, and altering tint. A particularcolor modification to indigo-dyed denim is fading to a “vintage look,”which has a less intense blue/violet tint and more subdued greyappearance than the freshly-dyed denim

As used herein, the term “bleaching” refers to the process of treating atextile material such as a fiber, yarn, fabric, garment or non-wovenmaterial to produce a lighter color. This term includes the productionof a brighter and/or whiter textile, e.g., in the context of a textileprocessing application, as well as lightening of the color of a stain,e.g., in the context of a cleaning application.

As used herein, the terms “size” and “sizing” refer to compounds used inthe textile industry to improve weaving performance by increasing theabrasion resistance and strength of a yarn. Size is usually made ofstarch or starch-like compounds.

As used herein, the terms “desize” and “desizing” refer to the processof eliminating/removing size (generally starch) from a textile, usuallyprior to applying special finishes, dyes or bleaches.

As used herein, the term “desizing enzyme(s)” refers to an enzyme usedto remove size. Exemplary enzymes are amylases, cellulases, andmannanases.

As used herein, the term “% identity” refers to the level of nucleicacid sequence identity between a nucleic acid sequence that encodes alaccase as described herein and another nucleic acid sequence, or thelevel of amino acid sequence identity between a laccase enzyme asdescribed herein and another amino aid sequence. Alignments may beperformed using a conventional sequence alignment program. Exemplarylevels of nucleic acid and amino acid sequence identity include, but arenot limited to, at least 60%, at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or even at least 99%, or more, sequence identity to a givensequence, e.g., the coding sequence for a laccase or the amino acidsequence of a laccase, as described herein.

Exemplary computer programs that can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at www.ncbi.nlm nih.gov/BLAST. Seealso, Altschul, et al., 1990 and Altschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, et al., 1997.)

An alignment of selected sequences in order to determine “% identity”between two or more sequences, may be performed using, for example, theCLUSTAL-W program in Mac Vector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

As used herein, the terms “chemical mediator” and “mediator” are usedinterchangeably to refer to a chemical compound that functions as aredox mediator to shuttle electrons between an enzyme exhibiting oxidaseactivity (e.g., a laccase) and a secondary substrate or electron donor.Such chemical mediators are also known in the art as “enhancers” and“accelerators.”

As used herein, the terms “draining” or “dropping” with respect to abath in which textile materials are present refers to fully or partiallyreleasing/emptying the solvent and reagents present in a bath. Draininga bath is typically performed between process steps such that thesolvent and reagents present in one processing step do not interferewith a subsequent processing step. Draining may be accompanied by one ormore rinse steps to further remove such the solvent and reagents.

As used herein, the terms “secondary substrate” and “electron donor” areused interchangeably to refer to a dye, pigment (e.g., indigo),chromophore (e.g., polyphenolic, anthocyanin, or carotenoid), or othersecondary substrate to and from which electrons can be shuttled by anenzyme exhibiting oxidase activity.

The following abbreviations/acronyms have the following meanings unlessotherwise specified:

EC enzyme commission

EDTA ethylenediaminetetraacetic acid

kDa kiloDalton

MW molecular weight

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt % weight percent

° C. degrees Centigrade

H₂O water

dH₂O or DI deionized water

dIH₂O deionized water, Milli-Q filtration

g or gm gram

μg microgram

mg milligram

kg kilogram

μL and μl microliter

mL and ml milliliter

mm millimeter

μm micrometer

M molar

mM millimolar

μM micromolar

U unit

sec and ″ second

min and ′ minute

hr hour

eq. equivalent

N normal

RTU ready-to-use

U Unit

owg on weight of goods

CIE International Commission on Illumination

Numeric ranges are inclusive of the numbers defining the range. Thesingular articles “a,” “an,” “the,” and the like, include the pluralreferents unless otherwise clear from context. Unless otherwisespecified, polypeptides are written in the standard N-terminal toC-terminal direction and polynucleotides are written in the standard 5′to 3′ direction. It is to be understood that the particularmethodologies, protocols, and reagents described, are not intended to belimiting, as equivalent methods and materials can be used in thepractice or testing of the present compositions and methods. Althoughthe description is divided into sections to assist the reader, sectionheading should not be construed as limiting and the description in onesection may apply to another. All publications cited herein areexpressly incorporated by reference.

Laccase and Laccase Related Enzymes

The enzymatic oxidation systems, compositions, and methods include oneor more laccases or laccase-related enzymes, herein collectivelyreferred to as “laccases” or “laccase enzymes.” Such laccases includeany laccase enzyme encompassed by EC 1.10.3.2, according to theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). Laccase enzymes from microbial and plantorigin are known in the art. A microbial laccase enzyme may be derivedfrom bacteria or fungi (including filamentous fungi and yeasts).Suitable examples include a laccase derived or derivable from a strainof Aspergillus, Neurospora (e.g., N. crassa), Podospora, Botrytis,Collybia, Cerrena (e.g., C. unicolor), Stachybotrys, Panus (e.g., P.rudis), Thielavia, Fomes, Lentinus, Pleurotus, Trametes (e.g., T.villosa, and T. versicolor), Rhizoctonia (e.g., R. solani), Coprinus(e.g., C. plicatilis and C. cinereus), Psatyrella, Myceliophthora (e.g.,M. thermonhila), Schytalidium, Phlebia (e.g., P. radita (WO 92/01046)),or Coriolus (e.g., C. hirsutus (JP 2238885)), Spongipellis, Polyporus,Ceriporiopsis subvermispora, Ganoderma tsunodae, and Trichoderma.

A laccase may be produced by culturing a host cell transformed with arecombinant DNA vector that includes nucleotide sequences encoding thelaccase. The DNA vector may further include nucleotide sequencespermitting the expression of the laccase in a culture medium, andoptionally allowing the recovery of the laccase from the culture.

An expression vector containing a polynucleotide sequence encoding alaccase enzyme may be transformed into a suitable host cell. The hostcell may be a fungal cell, such as a filamentous fungal cell, examplesof which include but are not limited to species of Trichoderma [e.g., T.reesei (previously classified as T. longibrachiatum and currently alsoknown as Hypocrea jecorina], T. viride, T. koningii, and T. harzianum),Aspergillus (e.g., A. niger, A. nidulans, A. oryzae, and A. awamori),Penicillium, Humicola (e.g., H. insolens and H. grisea), Fusarium (e.g.,F. graminum and F. venenatum), Neurospora, Hypocrea, and Mucor. A hostcell for expression of a laccase enzyme may also be from a species ofCerrena (e.g., C. unicolor). Fungal cells may be transformed by aprocess involving protoplast formation and transformation of theprotoplasts followed by regeneration of the cell wall using techniquesknown in the art.

Alternatively, the host organism may from a species of bacterium, suchas Bacillus [e.g., B. subtilis, B. licheniformis, B. lentus, B. (nowGeobacillus) stearothermophilus, and B. brevis], Pseudomonas,Streptomyces (e.g., S. coelicolor, S. lividans), or E. coli. Thetransformation of bacterial cells may be performed according toconventional methods, e.g., as described in Maniatis, T. et al.,“Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor, 1982. Thescreening of appropriate DNA sequences and construction of vectors mayalso be carried out by standard procedures (cf. supra).

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells. In someembodiments, the expressed enzyme is secreted into the culture mediumand may be recovered therefrom by well-known procedures. For example,laccases may be recovered from a culture medium as described in U.S.Patent Publication No. 2008/0196173. In some embodiments, the enzyme isexpressed intracellularly and is recovered following disruption of thecell membrane.

In particular embodiments, the expression host may be Trichoderma reeseiwith the laccase coding region under the control of a CBH1 promoter andterminator (see, e.g., U.S. Pat. No. 5,861,271). The expression vectormay be, e.g., pTrex3g, as disclosed in U.S. Pat. No. 7,413,887. In someembodiments, laccases are expressed as described in U.S. PatentPublication Nos. 2008/0196173 or 2009/0221030.

The following laccase genes and laccases are described in U.S.Publication No. 2008/0196173:

A. Cerrena Laccase A1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 1):ATGAGCTCAA AGCTACTTGC TCTTATCACT GTCGCTCTCG TCTTGCCACT   50AGGCACCGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC  100AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCGGTACT AGCTGGGGGC  150ACATTCCCTG GAGCACTTAT TACCGGTCAG AAGGTATGGG AGATCAACTT  200GGTTGAATAG AGAAATAAAA GTGACAACAA ATCCTTATAG GGAGACAGCT  250TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG  300ACATCCATTG TGAGTATAAT TTAGGTCCGC TCTTCTGGCT ATCCTTTCTA  350ACTCTTACCG TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG  400GGCCGATGGT CCTGCCTTCG TTACTCAATG CCCTATCGTC ACCGGAAATT  450CCTTCCTGTA CGACTTTGAT GTTCCCGACC AACCTGGTAC TTTCTGGTAC  500CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGTG GCCCGTTCGT  550TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG  600GTATGTGCAT CATCATAGAG ATATAATTCA TGCAGCTACT GACCGTGACT  650GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC  700TCGCAAGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTCA CTTATAGTGG  750TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG  800TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA  850ACGTTAAACG CGGCAAACGG TATGTTATTG AACTCCCGAT TTCTCCATAC  900ACAGTGAAAT GACTGTCTGG TCTAGTTATC GATTTCGTCT GGTCTCCATC  950TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATTC 1050AAATCTTCGC AGGCCAACGA TACTCGTTCG TCGTAAGTCT CTTTGCACGA 1100TTACTGCTTC TTTGTCCATT CTCTGACCTG TTTAAACAGC TCCATGCCAA 1150CCGTCCTGAA AACAACTATT GGATCAGGGC CAAACCTAAT ATCGGTACGG 1200ATACTACCAC AGACAACGGC ATGAACTCTG CCATTCTGCG ATACAACGGC 1250GCACCTGTTG CGGAACCGCA AACTGTTCAA TCTCCCAGTC TCACCCCTTT 1300GCTCGAACAG AACCTTCGCC CTCTCGTGTA CACTCCTGTG GTATGTTTCA 1350AAGCGTTGTA ATTTGATTGT GGTCATTCTA ACGTTACTGC GTTTGCATAG 1400CCTGGAAACC CTACGCCTGG CGGCGCCGAT ATTGTCCATA CTCTTGACTT 1450GAGTTTTGTG CGGAGTCAAC ATTCGTAAAG ATAAGAGTGT TTCTAATTTC 1500TTCAATAATA GGATGCTGGT CGCTTCAGTA TCAACGGTGC CTCGTTCCTT 1550GATCCTACCG TCCCCGTTCT CCTGCAAATT CTCAGCGGCA CGCAGAATGC 1600ACAAGATCTA CTCCCTCCTG GAAGTGTGAT TCCTCTCGAA TTAGGCAAGG 1650TCGTCGAATT AGTCATACCT GCAGGTGTCG TCGGTGGACC TCATCCGTTC 1700CATCTCCATG GGGTACGTAA CCCGAACTTA TAACAGTCTT GGACTTACCC 1750GCTGACAAGT GCATAGCATA ACTTCTGGGT CGTGCGAAGT GCCGGAACCG 1800ACCAGTACAA CTTTAACGAT GCCATTCTCC GAGACGTCGT CAGTATAGGA 1850GGAACCGGGG ATCAAGTCAC CATTCGTTTC GTGGTATGTT TCATTCTTGT 1900GGATGTATGT GCTCTAGGAT ACTAACCGGC TTGCGCGTAT AGACCGATAA 1950CCCCGGACCG TGGTTCCTCC ATTGCCATAT CGACTGGCAC TTGGAAGCGG 2000GTCTCGCTAT CGTATTTGCA GAGGGAATTG AAAATACTGC TGCGTCTAAT 2050TTAACCCCCC GTACGCGGTT TCCCTCACAT CCTGGAGCTA AGCAGCTTAC 2100TAACATACAT TTGCAGAGGC TTGGGATGAG CTTTGCCCGA AGTATAACGC 2150GCTCAGCGCA CAAAAGAAGG TTGCATCTAA GAAAGGCACT GCCATCTAAT 2200TTTTGTAACA AACAAGGAGG GTCTCTTGTA CTTTTATTGG GATTTCTTTC 2250TTGGGGTTTA TTGTTAAACT TGACTCTACT ATGTTTGGAA GACGAAAGGG 2300GCTCGCGCAT TTATATACTA TCTCTCTTGG CATCACCTGC AGCTCAATCC 2350TTCAACCACC TAA 2363 Translated protein sequence (SEQ ID NO: 2):MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG   50TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP  100AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP  150KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG  200PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ  250SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDSGMNSAI  300LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL  350DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK  400VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG  450TGDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNLTPQ  500AWDELCPKYN ALSAQKKLNP STT  523B. Cerrena Laccase A2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 3):ATGAGCTCAA AGCTACTTGC TCTTATTACT GTCGCTCTCG TCTTGCCACT   50AGGCACTGAC GCCGGCATCG GTCCTGTTAC CGACTTGCGC ATCACCAACC  100AGGATATCGC TCCAGATGGC TTCACCCGAC CAGCTGTACT GGCTGGGGGC  150ACATTCCCCG GAGCACTGAT TACCGGTCAG AAGGTATGGG AGATCGATTT  200CGTTGAATAG AGAAATACAA CTGAAAACAA ATTCTTATAG GGAGACAGCT  250TCCAAATCAA TGTCATCGAC GAGCTTACCG ATGCCAGCAT GTTGACCCAG  300ACATCCATTG TGAGTATAAT ATGGGTCCGC TCTTCTAGCT ATCCTTTCTA  350ACTCTTACCC TCTAGCATTG GCACGGCTTC TTTCAGAAGG GATCTGCGTG  400GGCCGATGGT CCTGCCTTCG TTACTCAATG TCCTATCGTC ACCGGAAATT  450CCTTCCTGTA CGACTTTGAT GTCCCCGACC AACCTGGTAC TTTCTGGTAC  500CATAGTCACT TGTCTACTCA ATATTGCGAT GGTCTTCGGG GCCCGTTCGT  550TGTATACGAT CCAAAGGATC CTAATAAACG GTTGTACGAC ATTGACAATG  600GTATGTGCAT CATCATAAAA ATATAATTCA TGCAGCTACT GACCGCGACT  650GATGCTGCCA GATCATACGG TTATTACCCT GGCAGACTGG TACCACGTTC  700TCGCACGAAC TGTTGTCGGA GTCGCGTAAG TACAGTCTGA CTTATAGTGG  750TCTTCTTACT CATTTTGACA TAGGACACCC GACGCAACCT TGATCAACGG  800TTTGGGCCGT TCTCCAGACG GGCCAGCAGA TGCTGAGTTG GCTGTCATCA  850ACGTTAAACG CGGCAAACGG TATGTCATTG AACTCCCGAT TTCTCCATTC  900ACATTGAAAT GACTGTCTGG TCTAGTTATC GATTCCGTCT GGTCTCCATC  950TCATGTGACC CTAATTACAT CTTTTCTATC GACAACCATT CTATGACTGT 1000CATCGAAGTC GATGGTGTCA ACACCCAATC CCTGACCGTC GATTCTATCC 1050AAATCTTCGC AGGCCAACGC TACTCGTTCG TCGTAAGTCT CTTTGAATGG 1100TTGGTGCTTT TTCTGTCCAT TCTCTAACCT GTTTATACAG CTCCATGCCA 1150ACCGTCCTGA AAACAACTAT TGGATCAGGG CCAAACCTAA TATCGGTACG 1200GATACTACCA CAGACAACGG CATGAACTCT GCCATTCTGC GATACAACGG 1250CGCACCTGTT GCGGAACCGC AAACTGTTCA ATCTCCCAGT CTCACCCCTT 1300TGCTCGAACA GAACCTTCGC CCTCTCGTGT ACACTCCTGT GGTATGTTTC 1350AAAGCGTTGT AATTTGATTG TGGTCATTCT AACGTTACTG CCTTTGCACA 1400GCCTGGAAAT CCTACGCCTG GCGGGGCCGA TATTGTCCAT ACTCTTGACT 1450TGAGTTTTGT GCGGAGTCAA CATTCGTAAA GATAAGAGTG TTTCTAATTT 1500CTTCAATAAT AGGATGCTGG TCGCTTCAGT ATCAACGGTG CCTCGTTCCT 1550TGATCCTACC GTCCCTGTTC TCCTGCAAAT TCTCAGCGGC ACGCAGAATG 1600CACAAGATCT ACTCCCTCCT GGAAGTGTGA TTCCTCTCGA ATTAGGCAAG 1650GTCGTCGAAT TAGTCATACC TGCAGGTGTT GTCGGTGGAC CTCATCCGTT 1700CCATCTCCAT GGGGTACGTA ACCCGAACTT ATAACAGTCT TGGACTTACC 1750CGCTGACAAG TGTATAGCAT AACTTCTGGG TCGTGCGAAG TGCCGGAACC 1800GACCAGTACA ACTTTAACGA TGCCATTCTC CGAGACGTCG TCAGTATAGG 1850AGGAACCGAG GATCAAGTCA CCATTCGATT CGTGGTATAT ACTTCATTCT 1900TGTGGATGTA TGTGCTCTAG GATACTAACT GGCTTGCGCG TATAGACCGA 1950TAACCCCGGA CCGTGGTTCC TCCATTGCCA TATCGACTGG CACTTGGAAG 2000CGGGTCTCGC TATCGTATTT GCAGAGGGAA TTGAAAATAC TGCTGCGTCT 2050AATCCAACCC CCCGTATGCG GTTTCCCACA CATTCTGAAT CTAAGCAGCT 2100TACTAATATA CATTTGCAGA GGCTTGGGAT GAGCTTTGCC CGAAGTATAA 2150CGCGCTCAAC GCACAAAAGA AGGTTGCATC TAAGAAAGGC ACTGCCATCT 2200AATCCTTGTA ACAAACAAGG AGGGTCTCTT GTACTTTTAT TGGGATTTAT 2250TTCTTGGGGT TTATTGTTCA ACTTGATTCT ACTATGTTTG GAAGTAGCGA 2300TTACGAAAGG GGCTTGCGCA TTTATATACC ATCTTTCTTG GCACCACCTG 2350CAGCTCAATC CTTCAACCAC CTAA 2374Translated protein sequence (SEQ ID NO: 4):MSSKLLALIT VALVLPLGTD AGIGPVTDLR ITNQDIAPDG FTRPAVLAGG   50TFPGALITGQ KGDSFQINVI DELTDASMLT QTSIHWHGFF QKGSAWADGP  100AFVTQCPIVT GNSFLYDFDV PDQPGTFWYH SHLSTQYCDG LRGPFVVYDP  150KDPNKRLYDI DNDHTVITLA DWYHVLARTV VGVATPDATL INGLGRSPDG  200PADAELAVIN VKRGKRYRFR LVSISCDPNY IFSIDNHSMT VIEVDGVNTQ  250SLTVDSIQIF AGQRYSFVLH ANRPENNYWI RAKPNIGTDT TTDNGMNSAI  300LRYNGAPVAE PQTVQSPSLT PLLEQNLRPL VYTPVPGNPT PGGADIVHTL  350DLSFDAGRFS INGASFLDPT VPVLLQILSG TQNAQDLLPP GSVIPLELGK  400VVELVIPAGV VGGPHPFHLH GHNFWVVRSA GTDQYNFNDA ILRDVVSIGG  450TEDQVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIE NTAASNPTPQ  500AWDELCPKYN ALNAQKKLNP STT  523C. Cerrena Laccase B1 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 5):ATGTCTCTTC TTCGTAGCTT GACCTCCCTC ATCGTACTAG TCATTGGTGC   50ATTTGCTGCA ATCGGTCCAG TCACTGACCT ACATATAGTG AACCAGAATC  100TCGACCCAGA TGGTTTCAAC CGCCCCACTG TACTCGCAGG TGGTACTTTC  150CCCGGTCCTC TGATTCGTGG TAACAAGGTA CGCTTCATAA CCGCCCTCCG  200TAGACGTAGG CTTCGGCTGA CATGACCATC ATCTGTAGGG AGATAACTTT  250AAAATTAATG TGATTGACGA CTTGACAGAG CACAGTATGC TCAAGGCTAC  300GTCCATCGTA AGTCCCTGAT TAACGTTTCA CCTGGTCATA TCGCTCAACG  350TCTCGAAGCA CTGGCATGGG TTCTTCCAGA AGGGAACCAA CTGGGCCGAT  400GGCCCCGCCT TTGTCACCCA ATGTCCTATC ACATCAGGAA ACGCCTTCCT  450GTATGATTTC AACGTTCCGG ACCAAGCTGG TACTTTCTGG TACCACAGCC  500ATCTCTCTAC ACAGTATTGT GACGGTCTTC GTGGTGCCTT TGTCGTCTAT  550GATCCTAATG ATCCCAACAA GCAACTCTAT GATGTTGATA ACGGCAAGTT  600CCTTGCATAT TTCATTTCTA TCATATCCTC ACCTGTATTG GCACAGAAAG  650CACCGTGATT ACCTTGGCTG ATTGGTATCA TGCCCTTGCT CAGACTGTCA  700CTGGTGTCGC GTGAGTGACA AATGGCCCTC AATTGTTCAC ATATTTTCCT  750GATTATCATA TGATAGAGTA TCTGATGCAA CGTTGATCAA CGGATTGGGA  800CGTTCGGCCA CCGGCCCCGC AAATGCCCCT CTGGCGGTCA TCAGTGTCGA  850GCGGAATAAG AGGTCAGTTC CATAATTATG ATTATTTCCC GCGTTACTTC  900CTAACAATTA TTTTTGTATC CCTCCACAGA TATCGTTTCC GATTGGTTTC  950TATTTCTTGC GACCCTAACT TTATTTTCTC AATTGACCAC CACCCAATGA 1000CCGTAATTGA GATGGACGGT GTTAATACCC AATCTATGAC CGTAGATTCG 1050ATCCAAATAT TCGCAGGTCA ACGATATTCA TTTGTCGTAG GTTATTATAA 1100ACTGCCCACC GATCATCTCT CACGTAACTG TTATAGATGC AAGCCAACCA 1150ACCAGTTGGA AATTATTGGA TCCGCGCTAA ACCTAATGTT GGGAACACAA 1200CTTTCCTTGG AGGCCTGAAC TCCGCTATAT TACGATATGT GGGAGCCCCT 1250GACCAAGAAC CGACCACTGA CCAAACACCC AACTCTACAC CGCTCGTTGA 1300GGCGAACCTA CGACCCCTCG TCTATACTCC TGTGGTATGT TGTTCTCGTT 1350ACATATACCA AACCTAATAT GAAGACTGAA CGGATCTACT AGCCGGGACA 1400GCCATTCCCT GGCGGTGCTG ATATCGTCAA GAACTTAGCT TTGGGTTTCG 1450TACGTGTATT TCACTTCCCT TTTGGCAGTA ACTGAGGTGG AATGTATATA 1500GAATGCCGGG CGTTTCACAA TCAATGGAGC GTCCCTCACA CCTCCTACAG 1550TCCCTGTACT ACTCCAGATC CTCAGTGGTA CTCACAATGC ACAGGATCTT 1600CTCCCAGCAG GAAGCGTGAT CGAACTTGAA CAGAATAAAG TTGTCGAAAT 1650CGTTTTGCCC GCTGCGGGCG CCGTTGGCGG TCCTCATCCT TTTCACTTAC 1700ATGGTGTAAG TATCAGACGT CCTCATGCCC ATATTGCTCC GAACCTTACA 1750CACCTGATTT CAGCACAATT TCTGGGTGGT TCGTAGCGCC GGTCAAACCA 1800CATACAATTT CAATGATGCT CCTATCCGTG ATGTTGTCAG TATTGGCGGT 1850GCAAACGATC AAGTCACGAT CCGATTTGTG GTATGTATCT CGTGCCTTGC 1900ATTCATTCCA CGAGTAATGA TCCTTACACT TCGGGTTCTC AGACCGATAA 1950CCCTGGCCCA TGGTTCCTTC ACTGTCACAT TGACTGGCAT TTGGAGGCTG 2000GGTTCGCTGT AGTCTTTGCG GAGGGAATCA ATGGTACTGC AGCTGCTAAT 2050CCAGTCCCAG GTAAGACTCT CGCTGCTTTG CGTAATATCT ATGAATTTAA 2100ATCATATCAA TTTGCAGCGG CTTGGAATCA ATTGTGCCCA TTGTATGATG 2150CCTTGAGCCC AGGTGATACA TGA 2173Translated protein sequence (SEQ ID NO: 6):MSLLRSLTSL IVLVIGAFAA IGPVTDLHIV NQNLDPDGFN RPTVLAGGTF   50PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF  100VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND  150PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA  200NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM  250TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY  300VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG  350FNAGRFTING ASLTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE  400IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN  450DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW  500NQLCPLYDAL SPGDT  515D. Cerrena Laccase B2 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 7):CACCGCGATG TCTCTTCTTC GTAGCTTGAC CTCCCTCATC GTACTAGCCA   50CTGGTGCATT TGCTGCAATC GGTCCAGTCA CCGACCTACA TATAGTGAAC  100CAGAATCTCG CCCCAGATGG TTTAAACCGC CCCACTGTAC TCGCAGGTGG  150TACTTTCCCC GGTCCTCTGA TTCGTGGTAA CAAGGTACGC TTCATAACCG  200CCCTCCGTAG ACGTAGGCTT CGGCTGACAT GACCATCATC TGTAGGGAGA  250TAACTTTAAA ATTAATGTGA TTGACGACTT GACAGAACAC AGTATGCTCA  300AGGCTACGTC CATTGTAAGT CCCTGATTAA CGTTTCACCT GGTCATATCG  350CTCAACGTCT CGAAGCACTG GCATGGGTTC TTCCAGAAGG GAACCAACTG  400GGCCGATGGC CCCGCCTTTG TCACCCAATG TCCTATCACA TCAGGAAACG  450CCTTCTTGTA TGATTTCAAC GTTCCGGACC AAGCTGGTAC TTTCTGGTAC  500CACAGCCATC TCTCYACACA GTATTGTGAC GGTCTTCGTG GTGCCTTTGT  550CGTCTATGAT CCTAATGATC CCAACAAGCA ACTCTATGAT GTTGATAACG  600GCAAGTCCCT TGCATATTTC AGTTCTATCA TATCCTCACC TGTATTGGCA  650CAGAAAGCAC CGTGATTACC TTGGCTGATT GGTATCATGC CCTTGCTCAG  700ACTGTCACTG GTGTCGCGTG AGTGACAAAT GGCCCTTAAT TGTTCACATA  750TTTTCCTGAT TATCATATGA TAGAGTATCT GATGCAACGT TGATCAACGG  800ATTGGGACGT TCGGCCACCG GCCCCGCAAA TGCCCCTCTG GCGGTCATCA  850GTGTCGAGCG GAATAAGAGG TCAGTTCCAT AATTATGATT ATTTCCCGCG  900TTACTTCCTA ACGATTATTT TTGTATCCCT CCACAGATAT CGTTTCCGAT  950TGGTTTCTAT TTCTTGCGAC CCTAACTTTA TTTTCTCAAT TGACCACCAC 1000CCAATGACCG TAATTGAGAT GGACGGTGTT AATACCCAAT CTATGACCGT 1050AGATTCGATC CAAATATTCG CAGGTCAACG ATATTCATTT GTCGTAGGTT 1100ATTATAAACT GCCCACCGAT CATCTCTCAC GTAACTGTTA TAGATGCAAG 1150CCAACCAACC AGTTGGAAAT TATTGGATCC GYGCTAAACC TAATGTTGGG 1200AACACAACTT TCCTTGGAGG CCTGAACTCC GCTATATTAC GATATGTGGG 1250AGCCCCTGAC CAAGAACCGA CCACTGACCA AACACCCAAC TCTACACCGC 1300TCGTCGAGGC GAACCTACGT CCCCTCGTCT ATACTCCTGT GGTATGTTGT 1350TCTCGTTACA TATACCAAAC CTAATATGAG GACTGAACGG ATCTACTAGC 1400CGGGACAGCC ATTCCCTGGC GGTGCTGATA TCGTCAAGAA CTTAGCTTTG 1450GGTTTCGTAC GTGTATTTCA CTTCCCTTTT GGCAGTAACT GAGGTGGAAT 1500GTATATAGAA TGCCGGGCGT TTCACAATCA ATGGAACATC CTTCACACCT 1550CCTACAGTCC CTGTACTACT CCAGATCCTC AGTGGTACTC ACAATGCACA 1600GGATCTTCTT CCAGCAGGAA GCGTGATCGA ACTTGAACAG AATAAAGTTG 1650TCGAAATCGT TCTGCCCGCT GCGGGCGCCG TTGGCGGTCC TCATCCTTTC 1700CACTTACATG GTGTAAGTAT CAGACGTCCT CATGCCTATA TTGCTCCGAA 1750CCTTACACAC CTGATTTCAG CACAATTTCT GGGTGGTTCG TAGCGCCGGT 1800CAAACCACAT ACAATTTCAA TGATGCTCCT ATCCGTGATG TTGTCAGTAT 1850TGGCGGTGCA AACGATCAAG TCACGATCCG ATTTGTGGTA TGTATCTCGT 1900GCCTTGCATT CATTCCACGA GTAATGATCC TTACACTTCG GGTTCTCAGA 1950CCGATAACCC TGGCCCATGG TTCCTTCACT GTCACATTGA CTGGCATTTG 2000GAGGCTGGGT TCGCTGTAGT CTTTGCGGAG GGAATCAATG GCACTGCAGC 2050TGCTAATCCA GTCCCAGGTA AGACTCTCGC TGCTTTGCGT AATATCTATG 2100AATTTAAAGC ATATCAATTT GCAGCGGCTT GGAATCAATT GTGCCCGTTG 2150TATGATGCCT TGAGCCCAGG TGATACATGA TTACTCGTAG CTGTGCTTTC 2200TTATACATAT TCTATGGGTA TATCGGAGTA GCTGTACTAT AGTATGTACT 2250ATACTAGGTG GGATATGYTG ATGTTGATTT ATATAATTTT GTTTGAAGAG 2300TGACTTTATC GACTTGGGAT TTAGCCGAGT ACATACTGAT CTCTCACTAC 2350AGGCTTGTTT TGTCTTTGGG CGCTTACTCA ACAGTTGACT GTTTTTGCTA 2400TTACGCATTG AACCGCATTC CGGTCYGACT CGTGTCCTCT ACTGTGACTT 2450GTATTGGCAT TCTAGCACAT ATGTCTCTTA CCTATAGGAA CAATATGTCT 2500CAACACTGTT CCAAAACCTG CGTAAACCAA ATATCGTCCA TCAGATCAGA 2550TCATTAACAG TGCCGCACTA ACCTAATACA CTGGCARGGA CTGTGGAAAT 2600CCCTATAAAT GACCTCTAGA CCGTGAGGTC ATTGCAAGGT CGCTCTCCTT 2650GTCAAGATGA CCC 2663 Translated protein sequence (SEQ ID NO: 8):MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGLN RPTVLAGGTF   50PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF  100VTQCPITSGN AFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND  150PNKQLYDVDN GNTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA  200NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM  250TVDSIQIFAG QRYSFVMQAN QPVGNYWIRA KPNVGNTTFL GGLNSAILRY  300VGAPDQEPTT DQTPNSTPLV EANLRPLVYT PVPGQPFPGG ADIVKNLALG  350FNAGRFTING TSFTPPTVPV LLQILSGTHN AQDLLPAGSV IELEQNKVVE  400IVLPAAGAVG GPHPFHLHGH NFWVVRSAGQ TTYNFNDAPI RDVVSIGGAN  450DQVTIRFVTD NPGPWFLHCH IDWHLEAGFA VVFAEGINGT AAANPVPAAW  500NQLCPLYDAL SPGDT  515E. Cerrena Laccase B3 Gene (Partial) from ATCC20013 Strain

Polynucleotide sequence (SEQ ID NO: 9):GTGGGGGCGG ATCCCTAACT GTTTCGAATC GGCACCGAAG TATGCAGGTG   50TGACGGAGAT GAGGCGTTTT TTCATCTTCC ACTGCAGTAT AAAATGTCTC  100AGGTAACGTC CAGCTTTTTG TACCAGAGCT ACCTCCAAAT ACCTTTACTC  150GCAAAGGTTT CGCGATGTCT CTTCTTCGTA GCTTGACCTC CCTCATCGTA  200CTAGCCACTG GTGCATTTGC TGCAATCGGT CCAGTCACTG ACCTACATAT  250AGTGAACCAG AATCTCGCCC CAGATGGTTT CAACCGCCCC ACTGTACTCG  300CAGGTGGTAC TTTCCCCGGT CCTCTGATTC GTGGTAACAA GGTACGCTTC  350ATAACCGCCC TCCGTAGACG TAGGCTTCGG CTGACATGAC CATCATCTGT  400AGGGAGATAA CTTTAAAATT AATGTGATTG ACGACTTGAC AGAACACAGT  450ATGCTCAAGG CCACGTCCAT TGTAAGTCCC TGATTAACGT TTCACCTGGT  500CATATCGCTC AACGTCTCGA AGCACTGGCA TGGGTTCTTC CAGAAGGGAA  550CCAACTGGGC CGATGGCCCC GCCTTTGTCA CCCAATGTCC TATCACATCA  600GGAAACTCCT TCCTGTATGA TTTCAACGTT CCGGACCAAG CTGGTACTTT  650CTGGTACCAC AGCCATCTCT CTACACAGTA TTGTGACGGT CTTCGTGGTG  700CCTTTGTCGT CTATGATCCT AATGATCCCA ACAAGCAACT CTATGATGTT  750GATAACGGCA AGTCCCTTGC ATATTTCATT TCTATCATAT CCTCACCTGT  800ATTGGCACAG AAAGCACCGT GATTACCTTG GCTGATTGGT ATCATGCCCT  850TGCTCAGACT GTCACTGGTG TCGCGTGAGT GACAAATGGC CCTCAATTGT  900TCACATATTT TCCTGATTAT CATATGATAG AGTATCTGAT GCAACGTTGA  950TCAACGGATT GGGACGTTCG GCCACCGGCC CCGCAAATGC CCCTCTGGCG 1000GTCATCAGTG TCGAGCGGAA TAAGAGGTCA GTTCCATAAT TATGATTATT 1050TCCCGCGTTA CTTCCTAACA ATTATTCTTG TATCCCTCCA CAGATATCGC 1100TTCCGATTGG TGTCTATTTC TTGCGACCCT AACTTTATTT TCTCAATTGA 1150TCACCACCCA ATGACCGTAA TTGAGATGGA CGGTGTTAAT ACCCAATCTA 1200TGACCGTAGA TTCGATCCAA ATATTCGCAG GTCAACGATA TTCATTTGTC 1250GTAGGTTATT ATAAACTGCC CACCGATCAT CTCTCACGTA ACTGTTATAG 1300ATGCAAGCCA ACCAACCRGT TGGAAATTAT TGGATCC 1337Translated protein sequence (SEQ ID NO: 10):MSLLRSLTSL IVLATGAFAA IGPVTDLHIV NQNLAPDGFN RPTVLAGGTF   50PGPLIRGNKG DNFKINVIDD LTEHSMLKAT SIHWHGFFQK GTNWADGPAF  100VTQCPITSGN SFLYDFNVPD QAGTFWYHSH LSTQYCDGLR GAFVVYDPND  150PNKQLYDVDN GKTVITLADW YHALAQTVTG VAVSDATLIN GLGRSATGPA  200NAPLAVISVE RNKRYRFRLV SISCDPNFIF SIDHHPMTVI EMDGVNTQSM  250TVDSIQIFAG QRYSFVMQAN QPVGNYWI  278F. Cerrena Laccase C Gene (Partial) from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 11):TGCAATCGGA CCGGTBGCTG ACCTTCACAT TACGGACGAT ACCATTGCCC   50CCGATGGTTT CTCTCGTCCT GCTGTTCTCG CTGGCGGGGG TTTCCCTGGC  100CCTCTCATCA CCGGAAACAA GGTAATGCCT AATGGTTGCG TCTTTGTTGG  150TGCTCTCATT CATCCACGAC ATTTTGTACC AGGGCGACGC CTTTAAACTC  200AATGTCATCG ATGAACTAAC GGACGCATCC ATGCTGAAGY CGACTTCCAT  250CGTAAGTCTC GCTGTATTGC TCCTTGAGCC ATTTCATTGA CTATAACTAC  300AACCAGCACT GGCATGGATT CTTCCAAAAG GGTACTAATT GGGCAGATGG  350TCCCGCTTTT GTGAACCAAT GCCCCATCAC CACGGGAAAC TCCTTCTTGT  400ACGACTTCCA GGTTCCTGAT CAAGCTGGTA AGCATGAGAT TACACTAGGA  450AAGTTTAATT TAATAACTAT TCAATCAGGA ACCTACTGGT ATCATAGTCA  500TTTGTCTACG CAATACTGTG ATGGTCTCAG AGGTGCATTC GTTGTCTACG  550ACCCTTCAGA TCCTCACAAG GATCTCTACG ACGTCGACGA CGGTGAGCTT  600TGCTTTTTTC ATTGGTATCC ATTATCGCTC ACGTGTCATT ACTGCGCCAC  650AGAAAGTACC GTCATCACTT TGGCTGATTG GTATCATACT TTGGCTCGTC  700AGATTGTTGG CGTTGCGTGA GTAGTCTTGT ACCGACTGAA ACATATTCCA  750GTTGCTGACT TCCCCACAGC ATTTCTGATA CTACCTTGAT AAACGGTTTG  800GGCCGCAATA CCAATGGTCC GGCTGATGCT GCTCTTGCTG TGATCAATGT  850TGACGCTGGC AAACGGTGTG TCCAGATTAC TATACTCCCC ATGACGTCTC  900AATGCTGATG TGTACTACTT CCAGGTACCG TTTCCGTCTT GTTTCCATAT  950CCTGTGACCC CAATTGGGTA TTCTCGATTG ACAACCATGA CTTTACGGTC 1000ATTGAAGTCG ATGGTGTTAA CAGTCAACCT CTCAACGTCG ATTCTGTTCA 1050GATCTTCGCC GGACAACGTT ACTCGTTCGT 1080Translated protein sequence (SEQ ID NO: 12):AIGPVADLHI TDDTIAPDGF SRPAVLAGGG FPGPLITGNK GDAFKLNVID   50ELTDASMLKX TSIHWHGFFQ KGTNWADGPA FVNQCPITTG NSFLYDFQVP  100DQAGTYWYHS HLSTQYCDGL RGAFVVYDPS DPHKDLYDVD DESTVITLAD  150WYHTLARQIV GVAISDTTLI NGLGRNTNGP ADAALAVINV DAGKRYRFRL  200VSISCDPNWV FSIDNHDFTV IEVDGVNSQP LNVDSVQIFA GQRYSF  246G. Cerrena Laccase D1 Gene from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 13):GATTCTAATA GACCAGGCAT ACCAAGAGAT CTACAGGTTG ACAGACCATT   50CTTCTAGGCG GCATTTATGC TGTAGCGTCA GAAATTATCT CTCCATTTGT  100ATCCCACAGG TCCTGTAATA ACACGGAGAC AGTCCAAACT GGGATGCCTT  150TTTTCTCAAC TATGGGCGCA CATAGTCTGG ACGATGGTAT ATAAGACGAT  200GGTATGAGAC CCATGAAGTC AGAACACTTT TGCTCTCTGA CATTTCATGG  250TTCACACTCT CGAGATGGGA TTGAACTCGG CTATTACATC GCTTGCTATC  300TTAGCTCTGT CAGTCGGAAG CTATGCTGCA ATTGGGCCCG TGGCCGACAT  350ACACATTGTC AACAAAGACC TTGCTCCAGA TGGCGTACAA CGTCCAACCG  400TGCTTGCCGG AGGCACTTTT CCTGGGACGT TGATCACCGG TCAGAAAGTA  450AGGGATATTA GTTTGCGTCA AAGAGCCAAC CAAAACTAAC CGTCCCGTAC  500TATAGGGTGA CAACTTCCAG CTCAATGTCA TCGATGATCT TACCGACGAT  550CGGATGTTGA CGCCAACTTC CATTGTGAGC CTATTATTGT ATGATTTATC  600CGAATAGTTT CGCAGTCTGA TCATTGGATC TCTATCGCTA GCATTGGCAC  650GGTTTCTTCC AGAAGGGAAC CGCTTGGGCC GACGGTCCCG CCTTCGTAAC  700TCAGTGCCCT ATAATAGCAG ATAACTCTTT TCTGTATGAC TTCGACGTCC  750CAGACCAAGC TGGTACTTTC TGGTATCATA GTCATCTATC CACTCAGTAC  800TGTGACGGTT TACGTGGTGC CTTCGTTGTG TACGATCCTA ACGATCCTCA  850CAAAGACCTA TACGATGTTG ATGACGGTGG GTTCCAAATA TTTGTTCTGC  900AGACATTGTA TTGACGGTGT TCATTATAAT TTCAGAGAGC ACCGTGATTA  950CCCTTGCGGA TTGGTACCAT GTTCTCGCCC AGACCGTTGT CGGCGCTGCG 1000TGAGTAACAC ATACACGCGC TCCGGCACAC TGATACTAAT TTTTTTTTAT 1050TGTAGCACTC CTGATTCTAC CTTGATCAAC GGGTTAGGCC GTTCACAGAC 1100CGGACCCGCT GATGCTGAGC TGGCTGTTAT CAGCGTTGAA CATAACAAAC 1150GGTATGTCAT CTCTACCCAG TATCTTCTCT CCTGCTCTAA TTCGCTGTTT 1200CACCATAGAT ACCGTTTCCG TTTGGTTTCG ATTTCGTGCG ACCCCAACTT 1250TACCTTCTCC GTTGATGGTC ATAATATGAC TGTCATCGAA GTCGATGGTG 1300TCAACACACG ACCCCTGACC GTTGACTCTA TTCAAATCTT CGCCGGACAG 1350AGGTATTCCT TTGTCGTAAG TTAATCGATA TATTCTCCTT ATTACCCCTG 1400TGTAATTGAT GTCAATAGCT CAATGCTAAC CAACCCGAAG ACAATTACTG 1450GATCCGTGCT ATGCCAAACA TCGGTAGAAA TACAACAACA CTGGACGGAA 1500AGAATGCCGC TATCCTTCGA TACAAGAATG CTTCTGTAGA AGAGCCCAAG 1550ACCGTTGGGG GCCCCGCTCA ATCCCCGTTG AATGAAGCGG ACCTGCGTCC 1600ACTCGTACCT GCTCCTGTGG TATGTCTTGT CGCGCTGTTC CATCGCTATT 1650TCATATTAAC GTTTTGTTTT TGTCAAGCCT GGAAACGCTG TTCCAGGTGG 1700CGCAGACATC AATCACAGGC TTAACTTAAC TTTCGTACGT ACACCTGGTT 1750GAAACATTAT ATTTCCAGTC TAACCTCTCT TGTAGAGTAA CGGCCTCTTC 1800AGCATCAACA ACGCCTCCTT CACTAATCCT TCGGTCCCCG CCTTATTACA 1850AATTCTGAGC GGTGCTCAGA ACGCTCAAGA TTTACTTCCA ACGGGTAGTT 1900ACATTGGCCT TGAACTAGGC AAGGTTGTGG AGCTCGTTAT ACCTCCTCTG 1950GCAGTTGGAG GACCGCACCC TTTCCATCTT CATGGCGTAA GCATACCACA 2000CTCCCGCAGC CAGAATGACG CAAACTAATC ATGATATGCA GCACAATTTC 2050TGGGTCGTCC GTAGTGCAGG TAGCGATGAG TATAACTTTG ACGATGCTAT 2100CCTCAGGGAC GTCGTRAGCA TTGGAGCGGG GACTGATGAA GTCACAATCC 2150GTTTCGTGGT ATGTCTCACC CCTCGCATTT TGAGACGCAA GAGCTGATAT 2200ATTTTAACAT AGACCGACAA TCCGGGCCCG TGGTTCCTCC ATTGCCATAT 2250TGATTGGCAT TTGGAGGCAG GCCTTGCCAT CGTCTTCGCT GAGGGCATCA 2300ATCAGACCGC TGCAGCCAAC CCAACACCCC GTACGTGACA CTGAGGGTTT 2350CTTTATAGTG CTGGATTACT GAATCGAGAT TTCTCCACAG AAGCATGGGA 2400TGAGCTTTGC CCCAAATATA ACGGGTTGAG TGCGAGCCAG AAGGTCAAGC 2450CTAAGAAAGG AACTGCTATT TAAACGTGGT CCTAGACTAC GGGCATATAA 2500GTATTCGGGT AGCGCGTGTG AGCAATGTTC CGATACACGT AGATTCATCA 2550CCGGACACGC TGGGACAATT TGTGTATAAT GGCTAGTAAC GTATCTGAGT 2600TCTGGTGTGT AGTTCAAAGA GACAGCCCTT CCTGAGACAG CCCTTCCTGA 2650GACAGCCCTT CCTGAGACGT GACCTCCGTA GTCTGCACAC GATACTYCTA 2700AATACGTATG GCAAGATGAC AAAGAGGAGG ATGTGAGTTA CTACGAACAG 2750AAATAGTGCC CGGCCTCGGA GAGATGTTCT TGAATATGGG ACTGGGACCA 2800 ACATCCGGA2809 Translated protein sequence (SEQ ID NO: 14):MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG   50TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP  100AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP  150NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG  200PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR  250PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI  300LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL  350NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK  400VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA  450GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ  500AWDELCPKYN GLSASQKVKP KKGTAI  526H. Cerrena Laccase D2 Gene from CBS115.075 Strain

Polynucleotide sequence (SEQ ID NO: 15):GATCTGGACG ATGGTATATA AGACGATGGT ATGAGACCCA TGAAGTCTGA   50ACACTTTTGC TCTCTGACAT TTCATGGTTC ATACTCTCGA GATGGGATTG  100AACTCGGCTA TTACATCGCT TGCTATCTTA GCTCTGTCAG TCGGAAGCTA  150TGCTGCAATT GGGCCCGTGG CCGACATACA CATTGTCAAC AAAGACCTTG  200CTCCAGATGG TGTACAACGT CCAACCGTGC TCGCCGGAGG CACTTTTCCT  250GGGACGTTGA TCACCGGTCA GAAAGTAAGG AATATTAGTT TGCGTCAAAG  300AGCCAACCAA AATTAACCGT CCCGTCCCAT AGGGTGACAA CTTCCAGCTC  350AATGTCATTG ATGATCTTAC CGACGATCGG ATGTTGACAC CAACTTCCAT  400TGTGAGCCTA TTATTGTATG ATTTATCCGT ATAGTTTCTC AGTCTGATCA  450TTGGCTCTCT ATCGCTAGCA TTGGCACGGT TTCTTCCAGA AGGGAACCGC  500TTGGGCCGAC GGTCCCGCCT TCGTAACTCA GTGCCCTATA ATAGCAGATA  550ACTCTTTTCT GTATGACTTC GACGTCCCCG ACCAAGCTGG TACTTTCTGG  600TATCATAGTC ATCTATCCAC TCAGTACTGT GACGGTTTAC GTGGTGCCTT  650CGTTGTGTAC GATCCTAACG ATCCTCACAA AGACCTATAC GATGTTGATG  700ACGGTGGGTT CCAAATACTT GACCAAGAAA CATTATATTG ATAGTATCCA  750CTCTGATTTT CAGAGAGCAC CGTGATTACC CTTGCGGATT GGTACCATGT  800TCTCGCCCAG ACCGTTGTCG GCGCTGCGTG AGTAACACAT ACACGCGCTC  850CGGCACACTG ATACTAATTT TTTATTGTAG CACTCCTGAT TCTACCTTGA  900TCAACGGGTT AGGCCGTTCA CAGACCGGAC CCGCTGATGC TGAGCTGGCT  950GTTATCAGCG TTGAACATAA CAAACGGTAT GTCATCTCTA CCCATTATCT 1000TCTCTCCTGC TTTAATTCGC TGTTTCACCA TAGATACCGA TTCCGTTTGG 1050TTTCGATTTC GTGCGACCCC AACTTTACCT TCTCCGTTGA TGGTCATAAT 1100ATGACTGTCA TCGAAGTCGA CGGTGTCAAC ACACGACCCC TGACCGTTGA 1150CTCTATTCAA ATCTTCGCCG GACAGAGGTA TTCCTTTGTC GTAAGTTAAT 1200CGATATATTC TCCCTATTAC CCCTGTGTAA TTGATGTCAA CAGCTCAATG 1250CTAACCAACC CGACGACAAT TACTGGATCC GTGCTATGCC AAACATCGGT 1300AGAAATACAA CAACACTGGA CGGAAAGAAT GCCGCTATCC TTCGATACAA 1350GAATGCTTCT GTAGAAGAGC CCAAGACCGT TGGGGGCCCC GCTCAATCCC 1400CGTTGAATGA AGCGGACCTG CGTCCACTCG TACCTGCTCC TGTGGTATGT 1450CTTGTCGTGC TGTTCCATCG CTATTTCATA TTAACGTTTT GTTTTTGTCA 1500AGCCTGGAAA CGCTGTTCCA GGTGGCGCAG ACATCAATCA CAGGCTTAAC 1550TTAACTTTCG TACGTACACC TGGTTGAAAC ATTATATTTC CAGTCTAACC 1600TCTTGTAGAG TAACGGCCTT TTCAGCATCA ACAACGCCTC CTTCACTAAT 1650CCTTCGGTCC CCGCCTTATT ACAAATTCTG AGCGGTGCTC AGAACGCTCA 1700AGATTTACTT CCAACGGGTA GTTACATTGG CCTTGAACTA GGCAAGGTTG 1750TGGAGCTCGT TATACCTCCT CTGGCAGTTG GAGGACCGCA CCCTTTCCAT 1800CTTCATGGCG TAAGCATACC ACACTCCCGC AGCCAGAATG ACGCAAACTA 1850ATCATGATAT GCAGCACAAT TTCTGGGTCG TCCGTAGTGC AGGTAGCGAT 1900GAGTATAACT TTGACGATGC TATCCTCAGG GACGTCGTGA GCATTGGAGC 1950GGGGACTGAT GAAGTCACAA TCCGTTTCGT GGTATGTCTC ACCCCTCGCA 2000TTTTGAGACG CAAGAGCTGA TATATTTTAA CATAGACCGA CAATCCGGGC 2050CCGTGGTTCC TCCATTGCCA TATTGATTGG CATTTGGAGG CAGGCCTTGC 2100CATCGTCTTC GCTGAGGGCA TCAATCAGAC CGCTGCAGCC AACCCAACAC 2150CCCGTACGTG ACACTGAGGG TTTCTTTATA GTGCTGGATT ACTGAATCGA 2200GATTTCTCCA CAGAAGCATG GGATGAGCTT TGCCCCAAAT ATAACGGGTT 2250GAGTGCGAGC CAGAAGGTCA AGCCTAAGAA AGGAACTGCT ATTTAAACG 2299Translated protein sequence (SEQ ID NO: 16):MGLNSAITSL AILALSVGSY AAIGPVADIH IVNKDLAPDG VQRPTVLAGG   50TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP  100AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP  150NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG  200PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR  250PLTVDSIQIF AGQRYSFVLN ANQPDDNYWI RAMPNIGRNT TTLDGKNAAI  300LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL  350NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK  400VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA  450GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ  500AWDELCPKYN GLSASQKVKP KKGTAI  526I. Cerrena Laccase E Gene (Partial) from CBS154.29 Strain

Polynucleotide sequence (SEQ ID NO: 17):TGCAATCGGA CCGGTGGCCG ACCTCAAGAT CGTAAACCGA GACATTGCAC   50CTGACGGTTT TATTCGTCCC GCCGTTCTCG CTGGAGGGTC GTTCCCTGGT  100CCTCTCATTA CAGGGCAGAA AGTACGTTAC GCTATCTCGG TGCTTTGGCT  150TAATTAAACT ATTTGACTTT GTGTTCTCTT AGGGGAACGA GTTCAAAATC  200AATGTAGTCA ATCAACTGAC CGATGGTTCT ATGTTAAAAT CCACCTCAAT  250CGTAAGCAGA ATGAGCCCTT TGCATCTCGT TTTATTGTTA ATGCGCCCAC  300TATAGCATTG GCATGGATTC TTCCAGAAGG GAACAAACTG GGCAGACGGT  350CCTGCGTTCG TGAACCAATG TCCAATCGCC ACGAACAATT CGTTCTTGTA  400TCAGTTTACC TCACAGGAAC AGCCAGGTGA GTATGAGATG GAGTTCATCC  450GAGCATGAAC TGATTTATTT GGAACCTAGG CACATTTTGG TACCATAGTC  500ATCTTTCCAC ACAATACTGC GATGGTTTGC GAGGGCCACT CGTGGTGTAT  550GACCCACAAG ACCCGCATGC TGTTCTCTAC GACGTCGACG ATGGTTCGTA  600CTTCGCATAT CCACGCTCGC TTTCATACAA TGTAAACTTT GTTCCTCCAG  650AAAGTACAAT CATCACGCTC GCGGATTGGT ATCATACCTT GGCTCGGCAA  700GTGAAAGGCC CAGCGTAAGG CACTTTAGTG TTTCCTCATA GTCCAAGAAA  750TTCTAACACG CCTTCTTCAT CAGGGTTCCT GGTACGACCT TGATCAACGG  800GTTGGGGCGT CACAACAATG GTCCTCTAGA TGCTGAACTA GCGGTGATCA  850GTGTTCAAGC CGGCAAACGG CAAGTTCAAT TCACACTTTT CACTCTGTAC  900CTTCTTCCTG ACATTCTTTT CTTGTAGTTA CCGCTTCCGC CTGATTTCAA  950TTTCATGCGA TCCCAACTAC GTATTCTCCA TTGATGGCCA TGATATGACT 1000GTCATCGAAG TGGATAGTGT TAACAGTCAA CCTCTCAAGG TAGATTCTAT 1050CCAAATATTT GCAGGTCAGA GATATTCGTT CGTGGTGAGT CAGATCAGGG 1100CATATCCTTT TGTCGATACG TCATTGACCA TATAATGCTA CAAGCTGAAT 1150GCCAACCAAC CAG 1163 Translated protein sequence (SEQ ID NO: 18):AIGPVADLKI VNRDIAPDGF IRPAVLAGGS FPGPLITGQK GNEFKINVVN   50QLTDGSMLKS TSIHWHGFFQ KGTNWADGPA FVNQCPIATN NSFLYQFTSQ  100EQPGTFWYHS HLSTQYCDGL RGPLVVYDPQ DPHAVLYDVD DESTIITLAD  150WYHTLARQVK GPAVPGTTLI NGLGRHNNGP LDAELAVISV QAGKRQVQFT  200LFTLYRFRLI SISCDPNYVF SIDGHDMTVI EVDSVNSQPL KVDSIQIFAG  250QRYSFVLNAN QP  262A Laccase D enzyme having the following amino acid sequence (SEQ ID NO:19; signal sequence in italics) may be used in the methods describedherein:

MGLNSAITSL AILALSVGSY AAIGPVADLH IVNKDLAPDG VQRPTVLAGG  50TFPGTLITGQ KGDNFQLNVI DDLTDDRMLT PTSIHWHGFF QKGTAWADGP 100AFVTQCPIIA DNSFLYDFDV PDQAGTFWYH SHLSTQYCDG LRGAFVVYDP 150NDPHKDLYDV DDGGTVITLA DWYHVLAQTV VGAATPDSTL INGLGRSQTG 200PADAELAVIS VEHNKRYRFR LVSISCDPNF TFSVDGHNMT VIEVDGVNTR 250PLTVDSIQIF AGQRYSFVLN ANQPEDNYWI RAMPNIGRNT TTLDGKNAAI 300LRYKNASVEE PKTVGGPAQS PLNEADLRPL VPAPVPGNAV PGGADINHRL 350NLTFSNGLFS INNASFTNPS VPALLQILSG AQNAQDLLPT GSYIGLELGK 400VVELVIPPLA VGGPHPFHLH GHNFWVVRSA GSDEYNFDDA ILRDVVSIGA 450GTDEVTIRFV TDNPGPWFLH CHIDWHLEAG LAIVFAEGIN QTAAANPTPQ 500AWDELCPKYN GLSASQKVKP KKGTAI 526

The mature processed form of this polypeptide is as follows (SEQ ID NO:20):

AIGPVADLHIVNKDLAPDGVQRPTVLAGGTFPGILITGQKGDNFQLNVIDDLTDDRMLTPTSIHWHGFFQKGTAWADGPAFVTQCPIIADNSFLYDFDVPDQAGTFWYHSHLSTQYCDGLRGAFVVYDPNDPHKDLYDVDDGGTVITLADWYHVLAQTVVGAATPDSTLINGLGRSQTGPADAELAVISVEHNKRYRFRLVSISCDPNFTFSVDGHNMTVIEVDGVNTRPLTVDSIQIFAGQRYSFVLNANQPEDNYWIRAMPNIGRNTTTLDGKNAAILRYKNASVEEPKTVGGPAQSPLNEADLRPLVPAPVPGNAVPGGADINHRLNLIFSNGLFSINNASFTNPSVPALLQILSGAQNAQDLLPTGSYIGLELGKVVELVIPPLAVGGPHPFHLHGHNFWVVRSAGSDEYNFDDAILRDVVSIGAGTDEVTIRFVTDNPGPWFLHCHIDWHLEAGLAIVFAEGINQTAAANPTPQAWDELCPKYNGLSASQKVKPK KGTAI

In some embodiments, laccase enzymes suitable for use in the presentcompositions and methods are mature polypeptides that lack a signalsequence that may be used to direct secretion of a full-lengthpolypeptide from a cell. A suitable mature polypeptide may have at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or even atleast 99%, or more, amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.Preferably, such polypeptides have enzymatic laccase activity, asdetermined using the assays and procedures described, herein.

In some embodiments, laccase enzymes suitable for use in the presentcompositions and methods are truncated with respect to a full-length ormature parent/reference sequence. Such truncated polypeptides may begenerated by the proteolytic degradation of a full-length or maturepolypeptide sequence or by engineering a polynucleotide to encode atruncated polypeptide. Exemplary polypeptides are truncated at the aminoand/or carboxyl-terminus with respect to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. The truncation maybe of a small number, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues, or of entire structural or functional domains. A suitabletruncated polypeptide may have at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or even at least 99%, or more, amino acidsequence identity to the corresponding portion of one or more of theabove-references amino acid sequences. Preferably, such polypeptideshave enzymatic laccase activity, as determined using the assays andprocedures described, herein.

Mediators

In some embodiments, the enzymatic oxidation systems, compositions, andmethods further include one or more chemical mediator agents thatenhance the activity of the laccase enzyme. A mediator (also called anenhancer or accelerator) is a chemical that acts as a redox mediator toeffectively shuttle electrons between the enzyme exhibiting oxidaseactivity and a dye, pigment (e.g., indigo), chromophore (e.g.,polyphenolic, anthocyanin, or carotenoid, for example, in a coloredstain), or other secondary substrate or electron donor.

In some embodiments the chemical mediator is a phenolic compound, forexample, methyl syringate, or a related compound, as described in, e.g.,PCT Application Nos. WO 95/01426 and WO 96/12845. The mediator may alsobe an N-hydroxy compound, an N-oxime compound, or an N-oxide compound,for example, N-hydroxybenzotriazole, violuric acid, orN-hydroxyacetanilide. The mediator may also be aphenoxazine/phenothiazine compound, for example,phenothiazine-10-propionate. The mediator may further be2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Otherchemical mediators are well known in the art, for example, the compoundsdisclosed in PCT Application No. WO 95/01426, which are known to enhancethe activity of a laccase. The mediator may also be acetosyringone,methyl syringate, ethyl syringate, propyl syringate, butyl syringate,hexyl syringate, or octyl syringate.

In some embodiments, the mediator is 4-cyano-2,6-dimethoxyphenol,4-carboxamido-2,6-dimethoxyphenol or an N-substituted derivative thereofsuch as, for example, 4-(N-methyl carboxamido)-2,6-dimethoxyphenol,4-[N-(2-hydroxyethyl) carboxamido]-2,6-dimethoxyphenol, or4-(N,N-dimethyl carboxamido)-2,6-dimethoxyphenol.

In some embodiments, the mediator is described by the following formula:

in which A is a group such as —R, -D, —CH═CH-D, —CH═CH—CH═CH-D, —CH═N-D,—N═N-D, or —N═CH-D, D is selected from the group consisting of —CO-E,—SO₂-E, —CN, —NXY, and —N⁺XYZ, E is —H, —OH, —R, —OR, or —NXY, and X, Y,and Z are independently selected from —H, —OH, —OR, and —R; where R is aC₁-C₁₆ alkyl, preferably a C₁-C₈ alkyl, which alkyl may be saturated orunsaturated, branched or unbranched and optionally substituted with acarboxy, sulfo or amino group; and B and C are independently selectedfrom C_(m)H_(2m+1); 1≦m≦5.

In some embodiments, A in the above mentioned formula is —CN or —CO-E,wherein E may be —H, —OH, —R, —OR, or —NXY, where X and Y areindependently selected from —H, —OH, —OR, and —R, where R is a C₁-C₁₆alkyl, preferably a C₁-C₈ alkyl, which alkyl may be saturated orunsaturated, branched or unbranched and optionally substituted with acarboxy, sulfo or amino group; and B and C are independently selectedfrom C_(m)H_(2m+1); 1≦m≦5. In some embodiments, the mediator is4-hydroxy-3,5-dimethoxybenzonitrile (also referred to as“syringonitrile” or “SN”).

Note that in the above mentioned formula, A may be placed meta to thehydroxy group, instead of being placed in the para position as shown.

For applications such as textile processing, the mediator may be presentin a concentration of about 0.005 to about 1.000 mmole per g denim,about 0.05 to about 500 mmole per g denim, about 0.1 to about 100 mmoleper g denim, about 1 to about 50 μmole per g denim, or about 2 to about20 μmole per g denim

The mediators may be prepared by methods known to the skilled artisan,such as those disclosed in PCT Application Nos. WO 97/11217 and WO96/12845 and U.S. Pat. No. 5,752,980. Other suitable mediators aredescribed in, e.g., U.S. Patent Publication No. 2008/0189871.

Methods of Use

The present systems and compositions can be use in applications whereenzymatic laccase activity is useful or desirable. Among theseapplications/methods is color modification of a substrate, which may beassociated with a textile. In some embodiments, such methods includeincubation of a laccase enzyme with a suitable substrate at a lowtemperature, for example, about 40° C. or less. In some embodiments, thetemperature is between about 20° C. and about 40° C. In someembodiments, the temperature is between about 20° to about 35° C. Insome embodiments, the temperature is about 20° C., 25° C., 30° C., or35° C. In some embodiments, the temperature is the ambient temperatureof tap water, for example, about 20° C. to about 23° C. The temperaturemay be maintained within a narrow range or allowed to fluctuate withoutsignificantly affecting the performance of the system and compositions.

The methods contemplate the use of one or more of the laccases describedherein. In some embodiments, the laccase is from a Cerrena species, suchas C. unicolor. In some embodiments, the laccase comprises, consists of,or consists essentially of the amino acid sequence of any of the C.unicolor laccase enzymes described herein, or an amino acid sequencehaving any of at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even99.5% identity to any of the C. unicolor laccase enzymes describedherein, and having laccase enzymatic activity.

In some embodiments, the systems and methods are used in a textileprocessing method, for example a method for modifying the color of atextile product, including, e.g., fibers, yarns, cloth, or completegarments. Generally, the methods involve contacting the textile with alaccase and a mediator for a length of time, and under conditions,sufficient to result in at least one (i.e., one or more) measurableeffects selected from, e.g., a change in color, a change in color cast,lightening, bleaching, fading, and/or a reduction ofredeposition/backstaining. In some embodiments, the methods are used toimpart a “vintage look” to dyed denim products. In the case ofindigo-dyed denim, the vintage look has a less intense blue/violet tintand more subdued grey appearance than the freshly-dyed denim. In thecase of sulfur-dyed denim, the vintage look is faded without the browntint that can result from hypochlorite treatment. Accordingly, while anaspect of the color modification obtained using laccases can becharacterized as a “bleaching” affect, this term does not fully describethe color modifications possible using laccases.

Textiles provided for color modification may be a cellulosic textiles orblends of cellulosic and synthetic fibers. In some embodiments, thetextile is denim dyed with indigo and/or a sulfur-based dye. In aparticular embodiment, the textile is dyed with indigo, and the laccaseenzyme and mediator are used to oxidize the indigo to isatin. The denimmay optionally be desized and/or stonewashed prior to color modificationwith the laccase enzyme.

Generally, given the same amount of abrasion in a textile processingmethod, denim strength is reduced to a greater degree at a highertemperature, compared to a lower temperature. Because the presentmethods can be performed at lower temperatures compared to conventionalmethods, they have the advantage of reducing the damage to textilesduring processing compared to conventional methods. Moreover, laccaseenzymes generally do not react with cellulosic textile fibers to reducetheir strength during processing. Accordingly, in some embodiments, thepresent methods do not affect the physical strength of the denim, orreduce the loss of physical strength compared to conventional methods.Where the denim is stretch denim comprising, e.g., elastane or spandex,and the present methods do not affect the stretch performance of thefabric, or reduce the loss of stretch performance compared toconventional methods.

In some embodiments, the laccase is used in a textile processing methodin combination with at least one other enzyme. Where such processing issimultaneous, enzymatic treatment may be performed at a low temperatureas described herein. Where the processing is sequential, the laccase maybe used at a low temperature as described herein, and the otherenzyme(s) may optionally also be used at a low temperature. In someembodiments, the laccase is used in combination with a cellulase enzyme,either simultaneously or sequentially. In one embodiment, the textile iscontacted with the laccase and cellulase simultaneously. In anotherembodiment, the textile is contacted with the laccase and cellulasesequentially. In one embodiment, the textile is contacted with thecellulase first to effect “stonewashing,” and then with the laccase toaffect color modification. In another embodiment, the textile iscontacted with the laccase first, and then with the cellulase. Wherecellulase and laccase treatments are sequential, the two processingsteps can be performed in the same bath, and without draining the bathbetween treatments. Such methods are referred to as “single-bath”methods.

Suitable cellulases may be derived from microorganisms which are knownto be capable of producing cellulolytic enzymes, such as, e.g., speciesof Humicola, Thermomyces, Bacillus, Trichoderma, Fusarium,Myceliophthora, Phanerochaete, Irpex, Scytalidium, Schizophyllum,Penicillium, Aspergillus or Geotricum. Known species capable forproducing celluloytic enzymes include Humicola insolens, Fusariumoxysporum or Trichoderma reesei. Non-limiting examples of suitablecellulases are disclosed in U.S. Pat. No. 4,435,307; European patentapplication No. 0 495 257; PCT Patent Application No. WO 91/17244; andEuropean Patent Application No. EP-A2-271 004, all of which areincorporated herein by reference.

In some embodiments, enzymatic “stonewashing” using a cellulase,bleaching using an aryl esterase, and color modification using alaccase, can be combined to provide a comprehensive enzymatic textileprocessing system. Such a system allows a textile processor to producetextiles with a wide variety of finishes without the need to useconventional textile processing chemical.

Laccases can also be used in other aspects of textile manufacturing,generally including aspects of treatment, processing, finishing,polishing, production of fibers, or the like. In addition to modifyingthe color of dyed denim, laccases can be used in de-coloring dyed waste(including indigo-dyed waste), in fabric dyeing, in textile bleachingwork-up, in fiber modification; in achieving enhanced fiber or fabricproperties, and the like.

In further embodiments, the present systems and compositions may also beused in a method for modifying the color of wool. For example, EuropeanPatent No. EP 0 504 005 discloses that laccases can be used for dyeingwool. Laccases can also be used in the leather industry. For example,laccases can be used in the processing of animal hides including but notlimited to de-hairing, liming, bating and/or tanning of hides.

The present systems and compositions may also be used in a method formodifying the color of pulp or paper products. Such methods involvecontacting the pulp or paper product in need of color modification witha laccase as described, herein, for a length of time and underconditions sufficient for color modification to occur. In particularembodiments, the color modification is bleaching.

The present systems and compositions may also be used in a method forhair color modification. Laccases have reportedly been found to beuseful for hair dyeing (see, e.g., WO 95/33836 and WO 95/33837). Suchmethods involve contacting the hair having a color to be modified withthe laccase for a length of time and under conditions suitable forchanging the color of the hair.

The present systems and compositions may also be used in the field ofwaste-water treatment. For example, laccases can be used indecolorization of colored compounds; in detoxification of phenoliccomponents; for anti-microbial activity (e.g., in water recycling); inbio-remediation; etc.

The present systems and compositions may also be used in thedepolymerization of high-molecular-weight aggregates, deinking wastepaper, the polymerization of aromatic compounds, radical-mediatedpolymerization and cross-linking reactions (e.g., paints, coatings,biomaterials), the activation of dyes, and coupling organic compounds.

The present systems and compositions may also be used in a cleaningcomposition or component thereof, or in a detergent for use in acleaning method. For example, laccases can be used in the cleaning,treatment or care of laundry items such as clothing or fabric; in thecleaning of household hard surfaces; in dish care, including machinedishwashing applications; and in soap bars and liquids and/or syntheticsurfactant bars and liquids. The enzymes presented herein can be useful,for example, in stain removal/de-colorization, and/or in the removal ofodors, and/or in sanitization, etc. Laccase mediators can be used assanitization and antimicrobial agents (e.g., wood protection,detergents), independently of or in conjunction with laccase enzymes.

Laccases can be used in other aspects of field of personal care. Forexample, laccases can be used in the preparation of personal productsfor humans such as fragrances, and products for skin care, hair care,oral hygiene, personal washing and deodorant and/or antiperspirants, forhumans. Laccases can be useful, for example, in hair dyeing and/orbleaching, nails dyeing and/or bleaching; skin dyeing and/or bleaching;surface modification (e.g., as coupling reagent); as an anti-microbialagent; in odor removal; teeth whitening; etc. Laccases can be used inthe field of contact lens cleaning. For example, laccases can be used inthe cleaning, storage, disinfecting, and/or preservation of contactlenses.

Laccases can be used in the field of bio-materials. For example,laccases can be used as bio-catalysts for various organic reactions;and/or in connection with biopolymers; in connection with packaging; inconnection with adhesives; in surface modification (activation andcoupling agent); in production of primary alcohols; in connection withbiosensors and/or organic syntheses; etc. Laccases are capable ofoxidizing a wide variety of colored compounds having different chemicalstructures, using oxygen as the electron acceptor.

The present systems and compositions may also be used for the removal oflignin from lignocellulose-containing material (e.g., thedelignification of pulp), the bleaching of lignocellulose-containingmaterial (i.e. the enzymatic de-inking of recycled paper) and/or thetreatment of waste water arising from the manufacture of paper orcellulose. Such processes may use a laccase enzyme in combination withadding or metering-in non-aromatic redox agents plus phenolic and/ornon-phenolic aromatic redox compounds, the phenolic and non-phenolicunits of the lignin either being oxidized directly by the action ofthese phenolic and/or non-phenolic aromatic compounds, or the ligninbeing oxidized by other phenolic and/or non-phenolic compounds producedby the oxidizing action of these compounds.

Laccases can be used in other aspects relating to pulp and paper. Forexample, laccases can be used in the manufacture of paper pulps andfluff pulps from raw materials such as wood, bamboo, and cereal ricestraw; the manufacture of paper and boards for printing and writing,packaging, sanitary and other technical uses; recycling of cellulosefiber for the purpose of making paper and boards; and the treatment ofwaste products generated by and treated at pulp or paper mills and otherfacilities specifically dedicated to the manufacture of paper, pulp, orfluff. Laccases can be useful, for example, in wood processing; in pulpbleaching; in wood fiber modification; in bio-glue (lignin activation)for MDF manufacturing; for enhanced paper properties; in ink removal; inpaper dyeing; in adhesives (e.g. lignin based glue for particle- orfiber boards); etc.

Laccases can be used in the field of feed. For example, the laccases canbe used as a feed additive alone or as part of a feed additive with theaim to increase the nutritional value of feed for any kind of animalssuch as chicken, cows, pigs, fish and pets; and/or as a processing aidto process plant materials and food industry by products with the aim toproduce materials/products suitable as feed raw materials.

Laccases can be used in the field of starch processing. For example,laccases can be used in the processing of a substrate including starchand/or grain to glucose (dextrose) syrup, fructose syrup or any othersyrup, alcohol (potable or fuel) or sugar. Such starch processing mayinclude processing steps such as liquefaction, saccharification,isomerization, and de-branching of a substrate.

Laccases can be used in the field of food. For example, laccases can beused in the preparation, processing, or as an active ingredient in foodssuch as yellow fat, tea based beverages, culinary products, bakery, andfrozen foods for human consumption. Laccases can be used, for example,as a bread improver, in food preservation, as an oxygen scavenger, etc.Laccases can be used for reducing or eliminating the microbial load ofvarious foods (e.g., meats) or feed.

Any of the methods or uses for laccases described herein may beperformed at a low temperature, e.g., at a temperature lower than about40° C., e.g., less than about 40° C., less than about 37° C., less thanabout 35° C., less than about 32° C., less than about 30° C., less thanabout 27° C., less than about 25° C., and less than about 22° C.Exemplary temperature ranges are from about 20° C. to less than about40° C. Exemplary temperatures are 20° C., 21° C., 22° C., 23° C., 24°C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33°C., 34° C., or 35° C. In some embodiments, the temperature is at roomtemperature or the ambient temperature of tap water, for example, about20° C. to about 23° C.

Any of the methods or uses for laccases described herein may beperformed using any of the laccase enzymes described herein, e.g.,laccases from Cerrena unicolor. In some embodiments, laccases are usedat a concentration of about 0.005 to about 5000 mg/liter, about 0.05 toabout 500 mg/liter, about 0.1 to about 100 mg/liter, or about 0.5 toabout 10 mg/liter. In some denim processing embodiments, a laccase isused at a concentration of about 0.005 to about 5000 mg/kg of denim,about 0.05 to about 500 mg/kg of denim, about 0.1 to about 100 mg/kg ofdenim, or about 0.5 to about 10 mg/kg of denim. In some embodiments, alaccase is used at a pH of about 5 to about 7, about 5.5 to about 6.5,about 5 to about 6, or about 6. Exemplary pH values are about 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, and 7.0.

Ready to Use Compositions and Kits

As described above, the present compositions include one or morelaccases, and optionally one or more mediators. In some embodiments, thecompositions comprise a polypeptide comprising, consisting of, orconsisting essentially of an amino acid sequence selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, or a variant or fragment, thereof. In particular embodimentsthe compositions comprise a polypeptide comprising, consisting of, orconsisting essentially of an amino acid sequence selected from SEQ IDNO: 19 and 20, or a variant or fragment, thereof. Preferably, suchpolypeptides have enzymatic laccase activity, which can be determinedusing the assays and procedures described, herein

Such composition can also be provided in the form of a “ready to use”(RTU) composition comprising, consisting of, or consisting essentiallyof a laccase enzyme and a mediator. In some embodiments, the mediator isselected from acetosyringone, syringaldehyde, syringamide, methylsyringamide, 2-hydroxyethyl syringamide, methyl syringate,syringonitrile, dimethylsyringamide, and syringic acid. In oneembodiment, the mediator is syringonitrile(4-hydroxy-3,5-dimethoxybenzonitrile). The RTU composition may furthercontain one or more compounds to provide a pH buffer when thecomposition is in solution. For example, in some embodiments, thecomposition contains monosodium phosphate and adipic acid as a bufferingsystem. The RTU composition may be in a solid, granular form for ease ofstorage and transportation. The composition is then diluted with waterto provide an aqueous solution for use, e.g., as described. RTUcompositions may also include any number of additional reagents, such asdispersants, surfactant, blockers, polymers, preservatives, and thelike.

The following examples are provided to illustrate the systems,compositions, and methods, and should in no way be construed aslimiting. Other aspects and embodiments will be apparent to the skilledperson in view of the description.

EXAMPLES

The following enzyme nomenclature is used in the Examples:

Trade name Description PRIMAGREEN ® EcoWhite 1 Mycobacterium smegmatisperhydrolase, S54V variant of SEQ ID NO: 1 PRIMAGREEN ® EcoFade LTCerrena unicolor laccase and syringonitrile in a dry formulationOPTISIZE ® 160 amylase Amylase from Bacillus amyloliquefaciens INDIAGE ®Neutra L Endoglucanase from Streptomyces sp. 11AG8 INDIAGE ® 2XLCellulase from Trichoderma reesei INDIAGE ® SUPER GX Cellulase fromTrichoderma reesei NOVOPRIME ® 268 Laccase from Aspergillus oryzaeNOVOPRIME ® F258 Methyl syringate DENILITE ® II S Laccase fromAspergillus oryzae and methyl syringate

Example 1 Effect of Temperature on Laccase-Mediated Color Modificationof Stonewashed Denim Enzyme

Granular Laccase D enzyme from Cerrena unicolor (38,000 U/g) was used inthis experiment. One laccase unit is defined as the amount of laccaseactivity that oxidizes 1 nmol of ABTS substrate per second underconditions of an assay based on the ability of laccase enzyme to oxidizeABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate)) into itscorresponding stable cation radical, ABTS⁺. Accumulation of the radicalcauses the ABTS to turn a dark green color and an increase in absorbanceat 420 nm. The color formation is proportional to laccase activity andis monitored against a laccase standard.

Mediator

4-hydroxy-3,5-dimethoxybenzonitrile (syringonitrile, SN) was purchasedfrom Punjab Chemicals & Crop Protection Limited (Mumbai, India).

Procedure

12 denim legs weighing approximately 3 kg (total) were desized in aUnimac UF 50 washing machine under the following conditions:

-   -   Desizing for 15 minutes at 10:1 liquor ratio 50° C. with 0.5 g/l        (15 g) of OPTISIZE® 160 amylase (Genencor) and 0.5 g/l (15 g) of        a non-ionic surfactant [e.g., Rucogen BFA (Rudolf Chemie) or        Ultravon RW (Huntsman)].    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Following desizing, the denim was stonewashed in a Unimac UF 50 washingmachine under the following conditions:

-   -   Cold rinse for 5 minutes at 10:1 liquor ratio.    -   Stonewashing for 60 minutes at 10:1 liquor ratio 55° C. with 1        kg of pumice stone, pH 4.5 (1 g/l tri-sodium citrate dihydrate        and 1 g/l citric acid monohydrate) and 1.2 g/l INDIAGE® 2XL        cellulase (Genencor).    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

After stonewashing, laccase treatment was performed in a Unimac UF 50washing machine according to the following process:

-   -   30 minutes at 10:1 liquor ratio, with either (i) C. unicolor        laccase D and syringonitrile at pH 6 (0.7 g/l monosodium        phosphate and 0.17 g/l adipic acid) and temperatures of 40° C.,        30° C., or 23° C. or (ii) NOVOPRIME® Base 268 and NOVOPRIME®        F258 at pH 4.8 (0.29 g/l monosodium phosphate and 0.56 g/l of        adipic acid) and temperatures of 40 or 30° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Evaluation of Denim Legs

The amount of color modification, reported as “bleaching,” of denim legswas evaluated after laccase treatment with a Minolta Chromameter CR 310in the CIE Lab color space with a D 65 light source. The CIE colorspace, also known as the CIELUV color space, was adopted by theInternational Commission on Illumination (CIE) in 1976, and involves thevalues L*, u*, and v* calculated as follows:

$L^{*} = {{116\left( \frac{Y}{Y_{0}} \right)^{1/3}} - 16}$${{when}\mspace{14mu} \frac{Y}{Y_{0}}} > 0.008856$u^(*) = 13L^(*)(u^(′) − u₀^(′)) v^(*) = 13L^(*)(v^(′) − v₀^(′))

-   -   where    -   Y: Tristimulus value Y (tristimulus value Y₁₀ can also be used.)    -   u′,v′: Chromaticity coordinates from the CIE 1976 UCS diagram    -   Y₀, u′₀, v′₀: Tristimulus value Y (or Y₁₀) and chromaticity        coordinates u′, v′ of the perfect reflecting diffuser.

For each denim leg, 8 measurements were taken and the results from the12 legs (96 measurements total) were averaged. The results are shown inTables 1 and 2 and in FIG. 1.

TABLE 1 Results using C. unicolor laccase and syringonitrile C. unicolorlaccase Syringonitrile Bleaching concentration, concentration, Temp.,level, Standard g/l (U/ml) g/l (mM) ° C. CIE* Lab deviation 0.54 0.07 4038.3/−1.2/−12.0 0.5/0.1/0.1 (20.5) (0.39) 0.3 0.07 40 37.6/−0.5/−12.30.6/0.1/0.1 (11.4) (0.39) 0.15 0.07 40 36.4/−0.2/−12.8 0.5/0.1/0.1 (5.7)(0.39) 0.54 0.07 30 36.2/−0.2/−12.8 0.5/0.1/0.1 (20.5) (0.39) 0.3 0.0730 36.1/−0.2/−13.0 0.5/0.1/0.1 (11.4) (0.39) 0.15 0.07 30 35.3/0.0/−13.30.5/0.1/0.1 (5.7) (0.39) 0.15 0.07 23 34.0/0.3/−13.5 0.6/0.1/0.1 (5.7)(0.39) (no steam)

TABLE 2 Results using A. oryzae laccase from and methyl syringateNOVOPRIME ® NOVOPRIME ® Bleaching Base 268 F258 conc., Temp., level,Standard conc., g/l g/l (mM) ° C. CIE* Lab deviation 0.47 0.07 4036.2/−0.5/ 0.6/0.1/0.2 (0.33) −11.2 0.27 0.07 40 36.5/−0.4/ 0.6/0.1/0.2(0.33) −11.4 0.15 0.07 40 35.7/−1.0/ 0.5/0.1/0.2 (0.33) −11.9 0.15 0.0730 33.9/0.1/ 0.5/0.1/0.2 (0.33) −12.6

The results show the effectiveness of C. unicolor laccase andsyringonitrile in affecting a color change of stonewashed denim

Example 2 Effect of the Laccase:Mediator Ratio on Color Modification ofStonewashed Denim Procedure

12 denim legs weighing approximately 3 kg (total) were desized andstonewashed as described in Example 1. After stonewashing, laccasetreatment was performed in a Unimac UF 50 washing machine according tothe following process:

-   -   C. unicolor laccase D and syringonitrile, 30 minutes at 10:1        liquor ratio, pH 6 (0.7 g/l monosodium phosphate and 0.17 g/l        adipic acid) at 40° C.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Evaluation of Denim Legs

Color modification of denim legs was evaluated as described inExample 1. The results are shown in Table 3 and FIG. 2.

TABLE 3 Results using C. unicolor laccase and syringonitrile indifferent ratios C. unicolor laccase Mediator Bleaching concentration,concentration, Temp., level, Standard g/l (U/ml) g/l (mM) ° C. CIE* Labdeviation 0.15 0.07 40 36.4/−0.2/−12.8 0.5/0.1/0.1 (5.7) (0.39) 0.150.08 40 37.0/−0.4/−12.7 0.5/0.1/0.1 (5.7) (0.44) 0.15 0.1 4037.1/−0.4/−12.7 0.6/0.1/0.1 (5.7) (0.55)

The results show that the ratio of laccase enzyme to mediator can bemanipulated to alter color modification.

Example 3 Effect of Temperature on Color Modification Performance ofComposition Containing Laccase and Mediator on Stonewashed Denim

For the purpose of investigating laccase-mediated color modificationperformance at low temperature, a “ready-to-use” (RTU) composition wasprepared as shown in Table 4. The monosodium phosphate and adipic acidprovide a buffering function at about pH 6 in an application of use asdescribed below.

TABLE 4 Ready-to-use formulation Component % w/w Monosodium phosphate(anhydrous) 70 Adipic acid 7 C. unicolor laccase D granules (38,000 U/g)15 Syringonitrile 8

Procedure

12 denim legs weighing approximately 3 kg (total) were desized andstonewashed as described in Example 1. After stonewashing, laccasetreatment was performed in a Unimac UF 50 washing machine according tothe following process:

-   -   30 minutes at 10:1 liquor ratio at 30° C. or without incoming        steam (i.e., temperature of 21-22° C.) with the RTU laccase        composition described above or DENILITE® II S (Novozymes) at        concentrations and temperatures as described in the Tables 5 and        6, below.]    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Evaluation of Denim Legs

Color modification of denim legs was evaluated as described inExample 1. The results are shown in Tables 5 and 6 and in FIGS. 3 and 4.

TABLE 5 Results using C. unicolor RTU composition RTU laccase, Temp.,Bleaching level, Standard % owg* ° C. CIE* Lab deviation 1 3035.1/−0.7/−13.5 0.6/0.1/0.2 3 30 38.5/−1.3/−12.6 0.7/0.1/0.2 1 21-2233.3/−0.4/−13.6 0.6/0.1/0.1 3 21-22 37.2/−1.013.3 0.7/0.1/0.1 *“owg” =on weight of goods

TABLE 6 Results using an A. oryzae laccase RTU composition DENILITE ®Temp., Bleaching level, Standard II S, % owg ° C. CIE* Lab deviation 330 36.1/−1.3/−10.9 0.6/0.1/0.2 3 21-22 33.8/−0.8/−12.1 0.5/0.1/0.2

The results show that a C. unicolor laccase RTU composition providessuperior color modification at low temperature compared to conventionalcommercial laccase compositions.

Example 4 One-Step Stonewashing and Color Modification at 30° C.

12 denim legs weighing approximately 3 kg (total) were desized in aUnimac UF 50 washing machine as described in Example 1.

Following desizing, the denim was stonewashed and bleached in a UnimacUF 50 washing machine under the following conditions:

-   -   30 minutes, 30° C. at 10:1 liquor ratio, pH 6, (i) 0.4% owg        INDIAGE® Super GX cellulase (Genencor)+3% owg RTU laccase        composition described in Example 3 (i.e.,        “stonewashing+bleaching 1-step”) or (ii) INDIAGE® Super GX        cellulase, alone (i.e., “stonewashing only”).    -   2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice        stones were used. The results are shown in Table 7 and FIG. 5.

TABLE 7 Results of one-step stonewashing and color modification Temp.Bleaching level, Standard (° C.) CIE*Lab deviation Stonewashing only 3025.4/1.3/−12 0.3/0.1/0.3 Stonewashing + bleaching, 1 step 3027.3/0.6/−12.2 0.5/0.2/0.2

The results show that color modification can be achieved using laccaseand cellulase simultaneously.

Example 5 Two-Step Stonewashing and Color Modification at 30° C.

12 denim legs weighing approximately 3 kg (total) were desized in aUnimac UF 50 washing machine as described in Example 1.

Following desizing, the denim was stonewashed in a Unimac UF 50 washingmachine under the following conditions:

-   -   30 minutes, 30° C. at 10:1 liquor ratio, pH 5.5, 0.4% owg        INDIAGE® Super GX cellulase (Genencor)

Following stonewashing, the denim was bleached in a Unimac UF 50 washingmachine under the following conditions:

-   -   30 minutes, 30° C. at 10:1 liquor ratio, pH 6, 3% owg RTU        laccase composition described in Example 3.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice        stones were used.

The results are shown in Table 8 and FIG. 5. The two-step stonewashingand color modification results were compared to the results forstonewashing alone as described in Example 4.

TABLE 8 Results of two-step stonewashing and color modification Temp.Bleaching level, Standard (° C.) CIE* Lab deviation Stonewashing only 3025.4/1.3/−12 0.3/0.1/0.3 Stonewashing + bleaching, 2 steps 3031.9/−0.3/−12.9 0.6/0.2/0.1

The results show that color modification by laccase treatment can beachieved following stonewashing.

Example 6 Laccase-Mediated Color Modification of Denim at 30° withoutStonewashing

12 denim legs weighing approximately 3 kg (total) were desized in aUnimac UF 50 washing machine as described in Example 1.

Following desizing, the denim was bleached in a Unimac UF 50 washingmachine under the following conditions:

-   -   30 minutes, 30° C. at 10:1 liquor ratio, pH 6, 3% owg RTU        laccase composition described in example 3.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio. No pumice        stones were used.

The results are shown in Table 9 and FIG. 5. The color modificationresults were compared to the results for stonewashing alone as describedin Example 4.

TABLE 9 Results of color modification without stonewashing Temp.Bleaching level, Standard (° C.) CIE* Lab deviation Stonewashing only 3025.4/1.3/−12 0.3/0.1/0.3 Bleaching, no stonewashing 30 26.9/0.7/12.10.5/0.1/0.2

The results show that the amount of color modification produced bylaccase treatment without stonewashing is higher than with stonewashingalone.

Example 7 Stonewashing and Color Modification with Cellulase and Laccasein a Single-Bath Bath Process without Pumice Stones

This Example shows that effective stonewashing and color modificationcan be obtained using laccase and cellulase in a single-bath process.

Enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).

Procedure

Starting material was desized denim weighing approximately 3 kg(ballast+2 legs for evaluation).

The denim was stonewashed in a Renzacci LX 22 washing machine under thefollowing conditions:

-   -   40 minutes, 50° C. at 10:1 liquor ratio, pH 6.5 0.4% owg of        INDIAGE® Neutra L cellulase (Batch No. 40105358001 activity 5197        NPCNU/g) (Genencor).    -   After stonewashing 1 leg was taken out and dried for evaluation.    -   Following stonewashing, and without draining (i.e., dropping)        the bath, the second denim leg was subjected to color        modification under the following conditions:    -   40 minutes, 40° C. at 10:1 liquor ratio and 1% owg of RTU        PRIMAGREEN® EcoFade LT 100    -   2 cold rinses for 3 minutes    -   The denim was dried in an industrial dryer

Evaluation of Denim Legs

Color modification and stonewashing on denim legs were evaluated afterlaccase treatment and after cellulase treatment with a MinoltaChromameter CR 310 in the CIE Lab color space with a D 65 light source.Six measurements were taken for each leg, and the results were averaged.

The results are summarized in Table 10. The amount of color modificationobtained with sequential (i.e., two-step) addition of cellulase andlaccase in a single bath was greater than that obtained by addingcellulase and laccase at the same time as in Example 4.

TABLE 10 Bleaching level, Standard CIE*Lab deviation Stonewashing27.8/1.1/−13.2 0.3/0.1/0.1 Stonewashing + bleaching, single bath34.7/0.0/−12.2 0.5/0.1/0.1

The results show that the amount of color modification obtained withsequential (i.e., two-step) addition of cellulase and laccase in asingle bath is greater than that obtained by adding cellulase andlaccase at the same time as in Example 4.

Example 8 Color Modification with Laccase and Pumice Stones

This Example shows that effective stonewashing and color modificationcan be obtained using pumice stones and a laccase-mediator system in asingle-bath process.

Enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 7809136160, 6,292GLacU/g).

Procedure

12 denim legs weighing approximately 3 kg (total) were desized in aUnimac UF 50 washing machine as described in Example 1.

Following desizing, the denim was stonewashed in a Unimac UF 50 washingmachine under the following conditions:

-   -   30 minutes, 30° C. at 10:1 liquor ratio, 3 kg of pumice stone,        with 3% PRIMAGREEN® EcoFade LT100 (Genencor). The blank/control        was performed only with stones in water.    -   2 cold rinses for 5 minutes at 30:1 liquor ratio.

Evaluation of Denim Legs

Color modification on denim legs were evaluated after laccase treatmentand after the stonewashing treatment with a Minolta Chromameter CR 310in the CIE Lab color space with a D 65 light source, as before. Theaverage of eight measurements taken on the outside of each leg werereported as the Bleaching level. The average of four measurements takenon the inside of each leg were reported as the Backstaining level.

The results are summarized in Tables 11 and 12.

TABLE 11 Bleaching level, Standard CIE*Lab deviation Stonewashing25.5/1.1/−11.4 0.3/0.1/0.2 Color modification 28.7/0.3/−12.0 0.6/0.1/0.1

TABLE 12 Backstaining level, Standard CIE*Lab deviation Stonewashing50.6/−1.2/−5.5 0.4/0.1/0.3 Stonewashing + color modification,52.2/−1.2/−4.0 0.4/0.1/0.3 single bath

The results show that laccase treatment provides color modification evenif pumice stones are present, and further shows reduction/removal ofbackstaining.

Example 9 Stonewashing and Color Modification of Sulphur Dyed Garments

The test garments were made of 100% cotton Twill fabric dyed withsulphur khaki brown dye. 21 garments weighing approximately 7 kg (total)were stonewashed in a 25 kg belly washer (36 rpm) under the followingconditions:

-   -   45 minutes, 55° C. at 18:1 liquor ratio, pH 4.5 at 1 g/l of        INDIAGE® 2XL    -   1 cold rinse for 3 minutes at 12:1 liquor ratio. No pumice        stones were used.    -   After washing the garments were dried for evaluation    -   3 garments (approximately 1 kg, total) stonewashed as described        above were treated with PRIMAGREEN® EcoFade LT 100 under the        following conditions:    -   15, 30 or 45 minutes, 40° C. at 50:1 liquor ratio and 1, 2 or 3        g/l of PRIMAGREEN® EcoFade LT 100. The blank/control was        performed with the garment washed for 15, 30 or 45 min with only        water.    -   1 cold rinse for 3 minutes.    -   The denim was dried in an industrial dryer.

Evaluation of Denim Legs

Color modification and stonewashing of sulphur dyed garments wereevaluated after laccase treatment and after the stonewashing treatmentwith a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65light source, as above. For each garment 10 measurements were taken andthe results were averaged.

The results are summarized in Tables 13

TABLE 13 Treatment CIE*Lab Before treatment 40.2/2.2/20.8 Whole complexcellulase 43.7/2.1/19.2 Blank, 15 min 44.9/1.3/18.3 Blank, 30 min46.1/1.3/19.1 Blank, 45 min 46.6/1.3/18.5 PRIMAGREEN ® Ecofade (1 g/l)15 min 45.1/2.9/16.5 PRIMAGREEN ® Ecofade (1 g/l) 30 min 45.5/3.3/16.8PRIMAGREEN ® Ecofade (1 g/l) 45 min 44.7/3.2/16.4 PRIMAGREEN ® Ecofade(2 g/l) 15 min 44.7/3.3/15.9 PRIMAGREEN ® Ecofade (2 g/l) 30 min45.3/3.5/15.9 PRIMAGREEN ® Ecofade (2 g/l) 45 min 45.0/3.5/15.6PRIMAGREEN ® Ecofade (3 g/l) 15 min 44.4/3.4/15.5 PRIMAGREEN ® Ecofade(3 g/l) 30 min 44.6/3.6/15.7 PRIMAGREEN ® Ecofade (3 g/l) 45 min45.0/3.6/15.4

The results show that the a and the b values of the color spacesignificantly change compared to the untreated fabric, as well as to theblank. The modification to the cast of the garments is visible by eye.

Example 10 Color Modification of Sulphur Dyed Garments withoutStonewashing

3 garments made of 100% cotton Twill fabric dyed with sulphur khakibrown dye and weighing approximately 1 kg (total) were treated in a 5 kgbelly washer (36 rpm) under the following conditions:

-   -   15, 30 or 45 minutes, 40° C. at 40:1 liquor ratio and 1, 2 or 3        g/l of PRIMAGREEN® EcoFade LT 100. The blank/control was        performed with the garment washed for 15, 30 or 45 min with just        water.    -   1 cold rinses for 3 minutes    -   The denim was dried in an industrial dryer

Evaluation of Denim Legs

Color modification and stonewashing on sulphur dyed garment wereevaluated after laccase treatment and after the stonewashing treatmentwith a Minolta Chromameter CR 310 in the CIE Lab color space with a D 65light source. For each garment 10 measurements were taken and theresults were averaged.

The results are summarized in Table 14

TABLE 14 Treatment CIE*Lab Before treatment 40.2/2.2/20.8  Blank 15 min41.1/2.0/19.7  Blank 30 min 41.9/2.1/20.4  Blank 45 min 42.4/2.1/20.29PRIMAGREEN ® Ecofade (1 g/l) 15 min 41.1/3.7/17.6  PRIMAGREEN ® Ecofade(1 g/l) 30 min 41.5/4.2/18.6  PRIMAGREEN ® Ecofade (1 g/l) 45 min41.6/4.0/18.1  PRIMAGREEN ® Ecofade (2 g/l) 15 min 40.4/3.9/17.0 PRIMAGREEN ® Ecofade (2 g/l) 30 min 41.2/4.2/17.3  PRIMAGREEN ® Ecofade(2 g/l) 45 min 41.6/4.3/17.3  PRIMAGREEN ® Ecofade (3 g/l) 15 min40.5/4.0/16.6  PRIMAGREEN ® Ecofade (3 g/l) 30 min 41.0/4.2/17.1 PRIMAGREEN ® Ecofade (3 g/l) 45 min 40.6/4.3/17.0 

The results show that the a and the b values of the color spacesignificantly change compared to the untreated fabric as well as to theblank. The modification to the cast of the garments is visible by eye.

Example 11 Stonewashing and Bleaching Performance with Cellulase andLaccase in a Single-Bath Process in the Presence of Surfactant andPumice Stone Enzyme

PRIMAGREEN® EcoFade LT 100 laccase (Batch No. 780913616, 6,292 GLacU/g).

Procedure

12 denim garments weighing 10 kg (total) and dyed with pure indigo weredesized in a Tupesa front loading machine (36 rpm) under the followingconditions:

-   -   10 minutes, 40° C. at 10:1 liquor ratio, pH 7, and 0.5 g/l of        lubricant, 0.2 g/l of dispersant (non ionic surfactant), and 0.2        g/l of polyester blocker (non ionic hydrophilic co-polymer).

Following desizing, the denim was de stonewashed under the followingconditions:

-   -   30 minutes, 47° C. at 5:1 liquor ratio, pH 6 with 7 kg of pumice        stones 4% owg of INDIAGE® Super GX cellulase (Genencor). 1        garment was taken out for evaluation    -   Following stonewashing, and without draining (dropping) the        bath, the denim was bleached under the following conditions:    -   30 minutes, 47° C. at 5:1 liquor ratio and 2% owg of RTU        PRIMAGREEN® EcoFade LT 100.    -   2 cold rinses for 2 minutes at 1:50 liquor ratio    -   The denim was dried in an industrial dryer

Evaluation of Denim Legs

Color modification and stonewashing on denim were evaluated afterlaccase treatment and after cellulase treatment with a MinoltaChromameter CR 310 in the CIE Lab color space with a D 65 light source.For each leg 8 measurements were taken and the results were averaged.

The results are summarized in Table 15.

Bleaching level Standard CIE*Lab deviation Stonewashing 27.6/0.6/−12.00.5/0.1/0.1 Stonewashing + bleaching single bath in 32.3/−0.1/−12.80.7/0.1/0.1 presence of surfactant and pumice stone

The results show that color modification by laccase treatment occurs inthe presence of pumice stones and in the presence of a surfactant.

The aspects, embodiments, and examples described herein are forillustrative purposes only. Various modifications will be apparent tothe skilled person, and are included within the spirit and purview ofthis application, and the scope of the appended claims. All publicationsand patent documents cited herein are hereby incorporated by referencein their entirety.

1. A textile processing method, comprising contacting a textile with alaccase enzyme and a mediator at a temperature less than 40° C., for alength of time and under conditions sufficient to cause a colormodification of the textile.
 2. The method of claim 2, wherein the colormodification is selected from lightening of color, change of color,change in color cast, reduction of redeposition/backstaining, andbleaching.
 3. The textile processing method of claim 1, wherein thetemperature is from about 20° C. to less than 40° C.
 4. The textileprocessing method of claim 1, wherein the temperature is from about 20°C. to about 30° C.
 5. The textile processing method of claim 1, whereinthe textile is indigo-dyed denim.
 6. The textile processing method ofclaim 1, wherein the textile is sulfur-dyed denim.
 7. The textileprocessing method of claim 1, wherein the denim is desized and/orstonewashed prior to or simultaneously with contacting the textile withthe laccase enzyme and the mediator.
 8. The textile processing method ofclaim 1, wherein the stonewashing and contacting the textile with thelaccase enzyme and the mediator occur in the same bath.
 9. The textileprocessing method of claim 1, further comprising contacting the textilewith a cellulase enzyme, simultaneously or sequentially with contactingthe textile with the laccase enzyme and the mediator.
 10. The textileprocessing method of claim 9, wherein contacting the textile with thecellulase enzyme and contacting the textile with the laccase enzyme andthe mediator are performed sequentially, and wherein contacting thetextile with the cellulase enzyme is performed prior to contacting thetextile with the laccase enzyme and the mediator.
 11. The textileprocessing method of claim 10, wherein contacting the textile with thecellulase enzyme and contacting the textile with the laccase enzyme andthe mediator are performed sequentially in the same bath withoutdraining the bath between contacting the textile with a cellulase enzymeand contacting the textile with the laccase enzyme and the mediator. 12.The textile processing method of claim 9, wherein contacting the textilewith the cellulase enzyme and contacting the textile with the laccaseenzyme and the mediator are performed a temperature less than 40° C. 13.The method of claim 1, wherein the laccase is a microbial laccase. 14.The method of claim 1, wherein the laccase is from a Cerrena species.15. The method of claim 1, wherein the laccase is from Cerrena unicolor.16. The method of claim 1, wherein the laccase is laccase D from C.unicolor.
 17. The method of claim 1, wherein the laccase has an aminoacid sequence that is at least 70% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:
 20. 18. The methodof claim 1, wherein the laccase has an amino acid sequence that is atleast 80% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 19, and SEQ ID NO:
 20. 19. The method of claim 1, whereinthe laccase has an amino acid sequence that is at least 70% identical toSEQ ID NO: 19 or SEQ ID NO:
 20. 20. The method of claim 1, wherein thelaccase has an amino acid sequence that is at least 80% identical to SEQID NO: 19 or SEQ ID NO:
 20. 21. The method of claim 1, wherein thelaccase has an amino acid sequence that is at least 90% identical to SEQID NO: 19 or SEQ ID NO:
 20. 22. The method of claim 1, wherein themediator is syringonitrile.
 23. The method of claim 1, wherein thetemperature is from about 20° to about 35° C.
 24. The method of claim 1,wherein the temperature is from about 20° C. to about 23° C.
 25. Themethod of claim 1, wherein the temperature is the ambient temperature oftap water.
 26. The method of claim 1, wherein the laccase enzyme and themediator are provided together in a ready-to-use composition.
 27. Themethod of claim 1, wherein the laccase enzyme and the mediator areprovided in a solid form.